Re: [Histonet] "Dirty" Immunos

[Elizabeth Chlipala via Histonet]

Paula

You need to sit down with them and review the slides together, you can't solve 
a problem without seeing the problem.  They need to explain and show you what 
they consider dirty.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Paula via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, August 10, 2017 8:24 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] "Dirty" Immunos

Hello,

Hoping we could get a recommendation on helping this issue..

We have a pathologist that complains about "dirty" stains every now and 
then..not all the time. What could this be/mean? Could it be debris, background 
staining?

We use the Bond III with their ready to use antibodies and their slides for 
tissue controls.  

 

Regarding the "dirty" complaint, could it come from our water baths, 
covertiles, paraffin, antibodies? 

 

I appreciate any feedback to help and I'll try anything.

Thank you,

Paula

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Re: [Histonet] Blood donations for money

[Elizabeth Chlipala via Histonet]

I would contact your local department of public health or your states governing 
agency for hazardous/biological waste - for example in Colorado our hazardous 
waste is controlled by the Colorado Department of Health.  You should be able 
to register a complaint through them.

On a personal note and no Bob I'm not calling you out.  Not all plasma donors 
are sleazy I personally have donated over 50 units of platelets where I used to 
work and was not paid for any of it.  I did get to watch TV or a movie for a 
couple hours since I donated during work hours (I would actually do paperwork 
when I was on the machine).  I would even donate for specific patients when 
necessary, apparently some patients did well with my platelets.  It was really 
fascinating, a small suburban 400 bed hospital with a very active blood bank, I 
learned a lot, it was a great experience overall.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Frazier, John via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Monday, July 24, 2017 11:58 AM
To: Bob Richmond
Cc: Histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Blood donations for money

Under FDA guidelines plasmapheresis donor are held to a higher standard than 
red cell downers when it comes to qualifications.
Plasmapheresis donors are required take a physical once a year by a registered 
nurse.
Each plasma donor center must a medical director that can only be filled by a 
licensed physician. They are required to be on site minimum 20 hours a month to 
review laboratory results donor records, physical reviews, and consult any 
donors that were rejected due to positive lab test results.
There are many more FDA, foreign government German Health Ministry, and 
internal company standards that each donor center must abide by. If not they 
can receive 483's which is a citation, warning letters or consent decree.
Each donor is required to donate a minimum of two times with negative test 
results before the units can be put into production. I'll plasma is tested for 
viral markers, total protein, AST(liver test), West Nile, parvovirus, sexually 
transmitted diseases and many other lab test. This lab tests are designed to 
not only monitor the integrity of the plasma pool but also the donor's health.
Prior to the plasma being put into production it goes through a series of 
detergents, cooling and heating, Ultraviolet light that will kill any viral, 
bacterial and or fungal material.
To my knowledge there has not been any diseases acquired by recipients of the 
pharmaceutical derivatives from plasma donors since the 1990's.

These donors may be paid but that is the only way to meet the huge supply of 
plasma needed to make all the different plasma based products. IVIG, RhoGam, 
CVM immunoglobulin, Albumin, *Alpha-1 Antitrypsin*

*Here is a link to the insight of plasma donations and the governing bodies*

http://www.donatingplasma.org/


from my iPhone



Sent from my iPhone
On Jul 23, 2017, at 12:51 PM, Bob Richmond  wrote:

Jorge A. Santiago-Blay, PhD asks about blood donation for money. I suppose he's 
in the US. I don't think there's any paid donation of whole blood in the US any 
more. This is probably a plasmapheresis center, where people donate twice a 
week. The red blood cells are returned to the donor. Two cycles of this are 
usually done at a session.

Many, though not all, plasma donors are pretty sleazy people. I'd ask the 
plasma center first, then complain to local authorities about it. Most of these 
plasma centers are franchise operations, and you could complain to their 
managers also.

Most plasma products (derivatives) can be sterilized so they don't transmit 
viruses. Or so we hope.

Bob Richmond
Samurai Pathologist
Maryville TN
**

Close to where I leave, there is an establishment for blood "donations".

Apparently, the establishment pays per donation. I hypothesize that the

money explains why the place is generally "hopping" (today, ca. 8:30AM,

there were ca, 25-30 cars parked in front of the establishment; Sunday

mornings, same story). Regularly, I see trash out of the store (incl. blood

splatter marks on the sidewalk, gauze, etc.).


Can someone tell me:

1. Where can one find information of the internal operations of

establishments like this?


2. Where can one report concerns about establishments like this?


3. More broadly, how can anyone *scientifically* tell whether the blood

"donated" at those (or any other) establishments is "safe" for use by other

humans?


Jorge A. Santiago-Blay, PhD

blaypublishers.com
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Re: [Histonet] heated block trimmers

[Elizabeth Chlipala via Histonet]

Fred

We have a block trimmer here in the lab it's not the newcomer supply its from 
Shandon, we like it.  I personally do not use it but the rest of the 
technicians do.   I'm a bit old school I have a very old steak knife that I 
have been using since 1996 so it's nice and dull and works perfectly for 
trimming excess paraffin off blocks.

What's nice about these trimmers is that they decrease the repetitive motion of 
scraping the block with a knife, so they are better overall.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Underwood, Fred via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, May 24, 2017 7:03 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] heated block trimmers

Hi All,

Anyone have any experience with the heated block trimmers?  Specifically,  I 
was looking at the one from Newcomer.

Thanks,
Fred
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Re: [Histonet] No Ribbon

[Elizabeth Chlipala via Histonet]

I might get some individuals upset but we really need to watch what we do in 
the histology lab and how that effects the precision of our process.  When you 
breath on the block you are basically generating a thicker section, it's not a 
good practice to keep.  Trust me I used to do this in the past.  Thicker 
sections especially for IHC will generate a more intense stain, most 
individuals may think that that should not be an issue but it is - in the 
clinical setting especially when you are staining for therapeutic markers such 
as Her2 and PD-L1 - 510K cleared kits for these recommend specific section 
thickness settings and in the research setting if you are running any type of 
analysis on a batch of samples.  If your microtome is set at 4 microns and you 
are breathing on the block you are not cutting at 4 microns, you are cutting 
thicker than that and a thicker section will increase staining intensity and 
therefore alter the results.

Just my two cents.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: Caroline Miller via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, April 11, 2017 11:59 AM
To: Paula Keene Pierce
Cc: Histonet Histonet
Subject: Re: [Histonet] No Ribbon

Yes, +1 to Paula, also:
- I often found they are too sharp (!!) to ribbon so a gentle wipe (in the 
direction of the bevel) with a kimwipe helps
- Also 'huffing' slightly on the block will warm the paraffin and both smooth 
out the wrinkles as well as make the sections stick together

:)

mills

On Tue, Apr 11, 2017 at 10:37 AM, Paula Keene Pierce via Histonet < 
histonet@lists.utsouthwestern.edu> wrote:

> Hi,
> the blades are packed with a tiny, bit of oil. This can cause you to 
> not obtain a ribbon.
> Try gently cleaning with xylene, followed by alcohol before 
> sectioning. Paula Keene Pierce, BS, HTL(ASCP)HTPresidentExcalibur 
> Pathology, Inc.5830 N Blue Lake DriveNorman, OK 73069PH 
> 405-759-3953FAX 405-759-7513www.excaliburpathology.com
>
>   From: Gareth Davis via Histonet 
> 
>  To: "Histonet@lists.utsouthwestern.edu"  utsouthwestern.edu>
>  Sent: Tuesday, April 11, 2017 11:55 AM
>  Subject: [Histonet] No Ribbon
>
> I feel like I should be able to figure this one out, but I have been 
> having trouble with getting a ribbon for a while now.  I have a Leica 
> Microtome
> RM2125 and I use either a AccuEdge blade (I get one block to cut then 
> it starts to compress) or a StatCut blade (sections do not stick 
> together at all), both High Profile.  The angle is about 4 microns.  I 
> change the angle a lot to see if that makes a difference and it 
> doesn't.  There are so many factors that could be the issue, I think 
> lack of humidity may be the main one. So, suggestions please!!
>
> --
> Ms. Gareth B. Davis, B.S., HT, QIHC (ASCP)cm Yuma Gastroenterology 
> Yuma, AZ 85364
> 928-248-5259
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>
>
>
> ___
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>



--
Caroline Miller (mills)
Director of Histology
3Scan.com
415 2187297
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Re: [Histonet] [EXTERNAL] Re: Tissue fixation

[Elizabeth Chlipala via Histonet]

I'm going to give my two cents here.  I think you need to look closely at how 
you handle these samples.  First of all leeps are not small samples therefore 
just because the leep has been placed in fixative upon removal and then sits in 
fixative until its processed.  Unless the sample is grossed  into smaller 
pieces only then it will adequately fix.   Electro cautery  instruments 
function to do two things, they cut and then cauterize - there is always a 
thermal defect associated with the process or thermal spread, this is clearly 
evident on the H slides.   The process is essentially coagulating the tissue 
proteins.  The thermal spread and char that is associated with this process 
varies dependent upon the instrument and setting that is being used.  This 
process may decrease fixative penetration so it's important that these samples 
are grossed in as soon as possible.  Trimming the tissue will allow for better 
penetration of the fixative and good fixation requires at least 4 to 6 hour
 s in formalin AFTER the tissue has been trimmed in.The artifact that the 
pathologist is seeing might not be related to the amount of time the sample 
sits in formalin it might be due to the amount of time the samples sits  in 
formalin prior to being grossed in.

If I had this issue I would start tracking the different times associated with 
these samples and possibly the instrument and setting used (if you are able to 
get that info).   

1.  Time to fixative - cold ischemic time
2.  Time to when the sample is grossed in 
3.  Time in formalin once the sample is grossed in

Good Luck I hope this helps.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Stedman, Nancy via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Friday, March 31, 2017 12:40 PM
To: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] [EXTERNAL] Re: Tissue fixation

I would bet there are quite a few pathologists on this mailing list (like me) 
who are here because we respect histo techs and know you have valuable 
information to offer!

I agree, cautery artifact is pretty easy to tell from poor fixation, so it 
should be easy to determine if that is the problem.  Sometimes, specimens that 
are submitted smashed in a cassette may have poor fixation of the tissue in 
contact with the cassette.  Or if they are submitted sponged and floating, some 
tissue may not be fully immersed in formalin.  Also, crush artifact may 
resemble poor fixation, so maybe the clinical team is not handling the tissue 
gently enough?  

I read veterinary specimens; we don't do LEEPs so I don't know if any of these 
scenarios are possible, but they are things I see frequently.  

-Nancy Stedman


-Original Message-
From: Mayer,Toysha N via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Friday, March 31, 2017 2:15 PM
To: 'histonet@lists.utsouthwestern.edu'
Subject: [EXTERNAL] Re: [Histonet] Tissue fixation

I agree with Rene.  To discredit the pathologist theory  show if all of the 
specimens from the run are not fixed properly.  Then show if it is just the 
LEEPs.  Then show if it is that particular clients specimens?  Then onto that 
client's LEEPs.
That should prove your problem lies with the client handling and not the 
fixation on your end.  Cauterization can cause burning of the specimen, but not 
look unfixed. If it was left out of formalin, autolysis can set in.
The situation shows the god-complex some physicians have and the lack of 
respect they have for their techs.  
This is why we should all be certified and keep up with continuing education, 
so that these pathologists will respect us.
Ok, I'm going to get off my soapbox now, before I say something ugly.

Toysha Mayer






--

Message: 3
Date: Fri, 31 Mar 2017 12:50:37 +
From: T H 
To: "histonet@lists.utsouthwestern.edu"

Subject: [Histonet] Tissue Fixation
Message-ID:



Content-Type: text/plain; charset="iso-8859-1"

Good Morning,


I have a pathologist that is not happy with the fixation on some of our LEEP 
specimens.  She swears its histology doing something to the specimen to cause 
the tissue to look unfixed on only "part" of the LEEP specimens (all the same 
client specimens).  She claims we must be diluting our formalin to cause this 
issue or "something".  We mentioned maybe it was on the clients end not placing 
them in 10% formalin right away, she wouldn't hear of it.


Let me give you some back ground on how our process works.  Our clients send us 
all our specimens to us via Overnight FedEx or 

Re: [Histonet] solvent recyclers - long response

[Elizabeth Chlipala via Histonet]

Bob

I believe that both the B/R an CBG recyclers are fractional distillation units, 
and to be honest I'm not even certain why I choose the CBG over B/R instrument 
when I purchased.  Prior to purchasing the CBG I was using the Suncycle 
Technologies system, these less expensive systems are more like filters and are 
not fractional distillation units, For alcohol percentage you get out what you 
put in.

We have been using the CBG recycler for many years now, we recycle our 95 and 
100 alcohol (I think it was around 2008 or so when we purchased it - we have a  
5 liter system) - adding lower percentage alcohols to the mix just decreases 
the alcohol percentage of the output.  We also recycle our xylene and propar.  
We do have defined SOP's that govern everything and we test the xylene for 
water content and record lot numbers for the xylene generated. The alcohol is 
checked with a hydrometer, adjusted for temp prior to making up lower 
percentage alcohols, and again we document with in house generated lot number  
for the lower grade alcohols we make.  We use the recycled xylene and alcohol 
in both the stainer and tissue processor, we even use recycled xylene in the 
coverslipper.  The only place we do not use recycled xylene is in the cleaning 
xylene station on the tissue processor we use new xylene for that.  Our 
instrument is serviced every other year, we are a low volume lab.  

Recycling has saved us money in reagents and in disposal, since we started 
recycling our waste classification changed from small quantity generator to 
conditionally exempt small quantity generator, so that saves us in additional 
fees for a state waste certificate and other regulatory documentation, 
training, etc.  that is required once you become a small quantity generator.  

In my opinion and I would say our clients opinions also, recycling has not 
affected the quality of our product, we process very small samples such as 
cartilage pellets up to large 2 x 3 slide research samples like lion vocal 
cords or portions of canine mandibles.  We have done extensive testing on the 
precision and accuracy of our IHC and H staining via image analysis and the 
quality of our processing, routine, special and IHC is consistent and has not 
been affected at all since we began recycling.   I firmly believe that you need 
to manage the reagents accordingly and make sure you follow the instructions 
and limitations of the instrument, for example you MUST not recycle any alcohol 
that has been used in deparaffinization.The documentation that we require 
does take some time but in our environment as GLP compliant lab it is required. 
 

There has been some extensive discussion on the value of recycling recently on 
the histonet and I would say that there are individuals that will not agree 
with my opinion, but based upon our experience it does works for us and I feel 
that it is of value personally.

On another note - Congrats on your retirement!!

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Bob Richmond via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Friday, March 24, 2017 12:01 PM
To: Histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] solvent recyclers

A few years ago the B/R spinning-band stills were definitely the still of 
choice for recovering histologic solvents, and the older generation of CBG 
machine were inadequate. I think though that the later generation of CBG stills 
predominates today. I don't know how they work. Could somebody give us an 
update on this subject?

Bob Richmond
Samurai Pathologist
Maryville TN
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Re: [Histonet] Recycled reagents in VIP processor

[Elizabeth Chlipala via Histonet]

We have been recycling for years now and have not had any adverse effects.  We 
have a VIP 3 and a VIP 6.  We do use fresh xylene for the cleaning cycle and we 
only recycle our 95 and absolute alcohols along with both xylene and propar.  
We use a fractional distillation unit from CBG.  We have a specific procedure 
that we follow so that each xylene cycle is issued a lot number and we test all 
alcohol with a hydrometer and adjust to temperature prior to using it to 
prepare, 50, 70, 80 and 95% solutions, all of those when prepared are given lot 
numbers.  If you are NSH members and on the BLOCK my SOP's for reagent control, 
etc. are in the library.  We have decreased our costs for both reagents and 
waste disposal to me its works quite well if you manage it properly.  We have 
moved from a small quantity generator to a conditionally exempt small quantity 
generator since we started recycling.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 881-0763 cell
(303) 682-9060 fax
l...@premierlab.com

Ship to address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

From: Margaryan, Naira via Histonet [histonet@lists.utsouthwestern.edu]
Sent: Wednesday, February 22, 2017 1:32 PM
To: Gareth Davis; histonet@lists.utsouthwestern.edu
Cc: naira.margar...@hsc.wvu.edu
Subject: Re: [Histonet] Recycled reagents in VIP processor

 WOULD LIKE TO KNOW AS WELL - IT IS WASTE OF MONEY

Thanks,
Naira


Ranked nationally in all 10 pediatric specialties by U.S. News & World Report  
(LCHOC Ver 1.0)


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From: Gareth Davis via Histonet [histonet@lists.utsouthwestern.edu]
Sent: Wednesday, February 22, 2017 2:23 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Recycled reagents in VIP processor

Hi,
I was always told not to use recycled reagents, i.e. Alcohol and Xylene, in
processors.  I am using a VIP 300, refurbished, and I would rather not use
recycled reagents in it.  But, during the last CAP inspection they
suggested I use the recycled to save money.  And now my administration
wants to cut cost.  Just wanted to know what labs were doing.
Thanks,


--
Ms. Gareth B. Davis, B.S., HT, QIHC (ASCP)cm
Yuma Gastroenterology
Yuma, AZ 85364
928-248-5259
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Re: [Histonet] Fibroblasts

[Elizabeth Chlipala via Histonet]

Bernice

There are lots of different markers for fibroblasts via IHC - If you are 
looking for a good source Acris Antibodies has quite a few fibroblast markers

Vimentin, 
Smooth Muscle Actin
FSP-1 (fibroblast specific protein)
Pro Collagen I 
CD34
Prolyl-4-Hydroxylase beta
HSP-47

I have a two page document that I will send to you that lists a lot of what I 
have already and what types of fibroblasts that they stain for - we have used 
most of these in various species.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: Bernice Frederick via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, January 04, 2017 1:02 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Fibroblasts

Hello all,
Don't have a ton of time to search so am asking. Any good markers specific to 
fibroblasts? And am drawing a blank for regular special stains for some reason. 
Researcher says Vimentin is not specific enough.
Thanks,
Bernice

Bernice Frederick HTL (ASCP)
Senior Research Tech
Pathology Core Facility
Robert. H. Lurie Cancer Center
Northwestern University
710 N Fairbanks Court
Olson 8-421
Chicago,IL 60611
312-503-3723
b-freder...@northwestern.edu

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Re: [Histonet] Grad Student Problem

[Elizabeth Chlipala via Histonet]

You can also thaw and refreeze we have done that before.  

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: Terri Braud via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, January 04, 2017 12:16 PM
To: 'histonet@lists.utsouthwestern.edu'
Cc: 'r...@psu.edu'
Subject: Re: [Histonet] Grad Student Problem

My response:
My first question is what temperature are the blocks that are too brittle? And 
at what temperature are you trying to cut?  Blocks stored in cryofreezers at 
-70o C or less are far too cold to cut without brittleness.  My suggestion 
would be to pull the blocks and put them into the cryostat at -18o C for at 
least 6 hours to acclimate to that temp, then try to cut and stain for IF.

If that doesn't work, then I would thaw in formalin and process as routine 
tissue for formalin fixed paraffin embedded.  The process that your student 
described in the 2nd step is not a complete, or valid process.
Depending on the T-Cell markers they are trying to demonstrate, one can 
successfully use standard IHC with appropriate clones, or with usually less 
success, use a modified IF stain procedure for FFPE sections. Since there are a 
"ba-zillion" T-cell markers, any further details are very dependent on the 
markers and the condition of the tissue.
Sincerely, Terri Braud

Terri L. Braud, HT(ASCP)
Anatomic Pathology Supervisor
Laboratory
Holy Redeemer Hospital
1648 Huntingdon Pike
Meadowbrook, PA 19046
ph: 215-938-3689
fax: 215-938-3874
Care, Comfort, and Heal
Today's Topics:

   1. grad student problem (Roberta Horner)
Message: 1
Date: Tue, 3 Jan 2017 18:05:15 +
From: Roberta Horner 
I got the following from a grad student here at Penn State. I am not sure how 
to solve his problem if possible. Does anyone have any suggestions I can 
forward?
Roberta Horner
Animal Diagnostic Lab
Penn State University

"I am having some difficulties sectioning mouse tumor samples for 
immunofluorescent analysis.  We originally went the OCT route because we are 
staining for T cell markers and were worried that the heating that occurs 
during paraffin embedding would compromise the T cell receptor.  The samples 
are a little old, but we are hoping to section and stain for immune cell 
infiltrates.  When sectioning with the cryostat, the tissue and OCT is quite 
brittle and the sample is not intact enough to transfer to a slide. Two 
colleagues have given the following suggestions:
1. Thaw the OCT blocks, remove the tissue, and place in a new block with fresh 
OCT media.  I've tried this technique on a few practice samples and the new OCT 
media seems to be less brittle and I'm able to get tissue on a slide, however 
the tissue itself still seems to be poor with either freeze or sectioning 
artifacts.
2. Thaw the OCT blocks in water, remove the tissue and place in formalin 
overnight, place in 70% EtOH, then paraffin embed.  Section on a microtome.  
Check the fluorescently labelled antibody data sheet to see if paraffin 
embedding interferes with binding.  Try to stain and see what happens.

I was hesitant to try the second suggestion because I have found no protocol 
that takes tissue originally stored in OCT blocks and subsequently redirects 
them to formalin and paraffin for microtome sectioning.  If you have any 
recommendations on how to move forward and section these difficult samples, or 
know anyone at the diagnostics lab or at Penn State that could help, that would 
be much appreciated!"

*


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Re: [Histonet] Macrophage Stain

[Elizabeth Chlipala via Histonet]

Jessica

We have not tried Ram-11 in sheep it's a macrophage antibody against rabbit, in 
our hands it did not cross react with human, cyno or rat.  We use MAC387 to 
stain macrophages in sheep.  It's not entirely specific to macrophages but it 
is what we have used in the past.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: Jessica Riggleman via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, October 18, 2016 11:08 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Macrophage Stain

Hello,

Could you please help me find a macrophage stain that will work with sheep 
(specifically sheep laminectomy sections)? We are thinking RAM-11 or HAM-56, 
but unsure.

Also, what about Rabbit PLF?

Please note both are embedded in mega cassettes for paraffin processing.

Thank you,
Jessica



_

Jessica Riggleman | Research Associate

Globus Medical, Inc.
Valley Forge Business Center
2560 General Armistead Avenue | Audubon, PA 19403
Ph: (610) 930-1800 ext. 2583 | Fax:

Confidentiality Note:  This email is confidential and intended solely for the 
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recipient, be advised that you have received this email in error and that any 
use, dissemination, forwarding, printing, or copying of this email is strictly 
prohibited. If you have received this email in error please contact the sender. 
Any views or opinions presented are solely those of the author and do not 
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attachments are believed to be free of any virus or other defects which might 
affect any computer or IT system into which they are received, no 
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arising in any way from the receipt or use thereof.


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Re: [Histonet] Cutting blocks for mouse tissue troubleshooting

[Elizabeth Chlipala via Histonet]

Blanca

If your samples are fixed and processed properly the sections should cut 
without wrinkles.  We process mouse pancreas on a 30 minute cycle - 30 minutes 
per station starting at 50% alcohol.  We trim and soak blocks on ice and we 
normally do not have issue with wrinkles.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: Blanca Lopez via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, October 05, 2016 8:06 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Cutting blocks for mouse tissue troubleshooting

Hello Histonets!
I  need to find out a better way to cut the whole pancreas mouse tissue without 
wrinkles, Does anybody has suggestion. I am having trouble to obtain a nice 
section without wrinkles. It is pretty big piece and crawl in on my embedding, 
is hard to stretch out without breaking. I cannot use ammonia water because 
they use the unstained for research and I don't want get discrepancies in any 
other stains.  Suggestions???
thanks

Blanca Lopez
Histotech (ASCP)
UTSW Tissue Resource K1.210
Simmons Comprehensive Cancer Center
UT Southwestern Medical Center
Telephone: 214-648-7598
Email: blanca.lo...@utsouthwestern.edu




UT Southwestern


Medical Center



The future of medicine, today.

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Re: [Histonet] Water for H Stainers?

[Elizabeth Chlipala via Histonet]

Paula

We have ours hooked up to tap water.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: P Sicurello via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, September 27, 2016 10:28 AM
To: HistoNet
Subject: [Histonet] Water for H Stainers?

Good Morning Everyone,



What type of water do you use with your automated H stainers?  House or 
deionized/distilled?



We cannot buy one of the waterless, fancy schmancy H stainers at this time.  
L Thank you in advance.

Sincerely,



Paula



Paula Sicurello, HTL (ASCP)CM

Histotechnology Specialist

UC San Diego Health

200 Arbor Drive

San Diego, CA 92103

(P): 619-543-2872



*Confidentiality Notice*: The information transmitted in this e-mail is 
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dissemination or other use of or taking of any action in reliance upon this 
information by persons or entities other than the intended recipient is 
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Re: [Histonet] Egg Whites and IHC Staining

[Elizabeth Chlipala via Histonet]

Jessica

There are a couple of things that we will try here when we have problems with 
tissue adherence

1.  We utilize a particular brand of plus slides  - trubond 380 - these have 
worked very well in our hands, we purchase them from Newcomer Supply
2.  Post fix in 10% NBF for 10 minutes after you run the slides down to water - 
this does not impact the H staining but can impact IHC staining we have seen 
that is may decrease sensitivity so you would need to potentially rework your 
IHC protocol
3.  Our Ram-11 IHC protocol requires HIER we have two different retrieval 
protocols that we have validated for this particular antibody - our routine 95C 
for 30 minutes and then a 70C for 2 hours - we use the longer lower temp 
retrieval when we have problems with tissue adherence, again you may need to 
potentially rework your IHC protocol for the lower temp retrieval.

Hope this helps, good luck

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Jessica Riggleman via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Monday, September 12, 2016 8:05 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Egg Whites and IHC Staining

Hello,

We are cutting large Rabbit PLF formalin fixed paraffin embedded sections.

We have had issues staining H and Goldner’s Trichrome. Egg whites is working 
as a great slide adhesive for these two, but not so much for IHC Staining (RAM 
11 specifically).

Any ideas/suggestions on how to get these sections to adhere? We have tried 
many methods, such as positively charged slides, chrom-alum gel, and so on.

Thank you for all input,
Jessica


_

Jessica Riggleman | Research Associate

Globus Medical, Inc.
Valley Forge Business Center
2560 General Armistead Avenue | Audubon, PA 19403
Ph: (610) 930-1800 ext. 2583 | Fax:

Confidentiality Note:  This email is confidential and intended solely for the 
use of the individual to whom it is addressed. If you are not the intended 
recipient, be advised that you have received this email in error and that any 
use, dissemination, forwarding, printing, or copying of this email is strictly 
prohibited. If you have received this email in error please contact the sender. 
Any views or opinions presented are solely those of the author and do not 
necessarily represent those of Globus Medical, Inc. Although this email and any 
attachments are believed to be free of any virus or other defects which might 
affect any computer or IT system into which they are received, no 
responsibility is accepted by Globus Medical, Inc. for any loss or damage 
arising in any way from the receipt or use thereof.


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Re: [Histonet] Movat Pentachrome

[Elizabeth Chlipala via Histonet]

If you are not that concerned over the alcian blue portion of the movats you 
can perform an elastic trichrome - that's what we do here.  Mordant like you 
would in Bouins, and rather than stain with weigerts hemastoxylin, stain with 
the elastic portion of the VVG, differentiate like you would for elastic fibers 
and then continue on with the rest of the trichrome, it turns out great.  I 
have an SOP if anyone wants it.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Forest Blankenship via Histonet 
[mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, September 01, 2016 11:24 AM
To: 'histonet@lists.utsouthwestern.edu'
Subject: Re: [Histonet] Movat Pentachrome

Angela, the Movat is a wonderful thing for showing  loss of elastic fibers in 
emphysema and cardiovascular cases. We ran a ton of them in training because 
our lab/histo school was affiliated with the Texas Heart Institute.
Currently at the hospital I work at we use a Kit(Polyscientific)  for the VVG 
part of the stain and Saffron du Galantis for the counterstain. It smells great 
when gently heated.
As far as remarks about a trichrome being able to show the same thing; yeah if 
you also run a VVG on a serial section to show elastic fibers  and you lose the 
awesomeness that is the Movat.

Forest Blankenship, HTL(ASCP)
Histology Manager
Driscoll Children's Hospital
Corpus Christ, TX

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Re: [Histonet] Gallocyanin - protocol

[Elizabeth Chlipala via Histonet]

We used the stain to stain nissl substance in mouse lumbar spine.

Purchased the dye from sigma - below is from the SDS Sheet

Product name : Gallocyanine
Product Number : 124508
Brand : Sigma-Aldrich
CAS-No. : 1562-85-2 

The procedure in Bancroft-Gamble uses Chrom Alum - which is chromium potassium 
sulfate as the mordant

Chrom Alum - 5 gm
dH20 - 100 ml
Gallocyanine - 50 mg

The chrome alum is dissolved in the distilled water, the gallocyanine is added 
and the solution slowly heated until it boils.  Boil for 5 minutes. Cool to 
room temp adjust the volume back to 100 ml.  Filter before use

The stain is easy - place in staining solution for 18-48 hours, rinse in water, 
dehydrate, clear and mount.   B states results are blue but If I remember 
correctly it turned out to be a bluish grey in color.  I tried to see if I had 
any images but I could not find any, we did this back in 2006 so it was some 
time ago.

If you search the histonet archives there is an older post of mine looking for 
some help on this stain, there is a nice response back from Dr. Kiernan 
explaining a bit more about the dye and staining.

Liz


Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Bob Richmond via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, August 18, 2016 6:02 PM
To: Histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Gallocyanin

Liz Chlipala at Premier Laboratory in Boulder CO writes:

>>We have run this stain [gallocyanin] before, purchased the reagent 
>>from
Sigma, it's pretty easy to do there is a procedure in Bancroft and Gamble.<<

What stain? What mordant (chrome, aluminum, none)? To stain what?

I think the person originally posting needs to extract this information from 
the person who wants the stain done.

Bob Richmond
Samurai Pathologist
Maryville TN
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Re: [Histonet] Gallocyanine

[Elizabeth Chlipala via Histonet]

We have run this stain before, purchased the reagent from Sigma, it's pretty 
easy to do there is a procedure in Bancroft and Gamble.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Bob Richmond via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, August 17, 2016 5:59 PM
To: Histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Gallocyanine

Eva Permaul at Georgetown University asks:

>>We just had a request for Gallocyanine staining. Does anyone do this? 
>>Can
you share your protocol? Where do you buy your reagents?<<

Gallocyanin (correct spelling - C.I. 51030) is a blue oxazine dye somewhat 
similar to celestin blue B. It's been used as a stain for Nissl substance.
Somebody at the AFIP was touting it as a hematoxylin substitute during the 
Great Hematoxylin Shortage of around 1972. I think it was used as a chrome alum 
lake for a nuclear stain. I've never seen it. You can get the dry dye from 
Sigma-Aldrich. John Kiernan's book and Bancroft & Stevens 4th ed both have 
formulas. But what does your client want to do with it?

Bob Richmond
Samurai Pathologist
Maryville TN
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Re: [Histonet] Antibodies storage tips

[Elizabeth Chlipala via Histonet]

We use freezer boxes and then record what is in the freezer boxes on a 
worksheet that is kept in an excel file.  The excel file has all of the boxes 
in it.



Here is an example of one of the pages


BOX 6 - Full





name

vendor

catalog #

lot #

date alqt

ul/aliquot

notes





AGE

Biorbyt

orb10064

A1268

5/7/14

25ul



CSPG (CS-56)

Sigma

C8035

032M4824

5/19/14

20ul

retest 2/2017

CSPG (CS-56)

Sigma

C8035

026M4834V

need aliq. (2x200ul); retest 10/2020

Alk Phos-ALPL

abcam

ab17973

GR157978-1

8/15/14

10ul



Osterix/Sp7

abcam

ab22552

GR138927-1

5/30/14

10ul

retest 5/28/19

Osterix/Sp7

abcam

ab22552

GR176974-2

3/5/15

10ul






















Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC

Premier Laboratory, LLC

PO Box 18592

Boulder, CO 80308

(303) 682-3949 office

(303) 682-9060 fax

(303) 881-0763 cell

l...@premierlab.com

www.premierlab.com



Ship to Address:



Premier Laboratory, LLC

1567 Skyway Drive, Unit E

Longmont, CO 80504





-Original Message-
From: Rene J Buesa via Histonet [mailto:histonet@lists.utsouthwestern.edu]
Sent: Tuesday, August 16, 2016 9:31 AM
To: Julio Benavides; histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Antibodies storage tips



I used to have several compartmented plastic alphabetized boxes with enough 
empty spaces to accommodate "new arrivals". If the spaces were used-up I just 
added a new box. Since they occupied several shelves, each shelf had the 
lettering identifying the boxes in each one.René



On Tuesday, August 16, 2016 6:29 AM, Julio Benavides via Histonet 
> 
wrote:



Hi all,



In our lab we carry out quite a number of IHCs for research purposes.

The number of mAbs and pAb stored in the frezeer is getting to the point that, 
when you need to do and IHC, you actually lose more time diving through the 
frezeer draws for the right vial than doing the IHC! is not a question than we 
are running our of freezer room, it is more to the point of having a proper and 
standarized way to keep the antibodies and than produce a proper registry of 
what we have and where to find it.



I was wondering, in an attempt to avoid reinventing the wheel, if you could be 
as kind as to share your tips for storing and organizing the antibodies 
(aliquots, stock solutions, intermediate dilutions) so chaos does not take 
over the lab!



As always, thank you very much for your time and advice!



Cheers



Julio





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Re: [Histonet] Caspase 3 and Tunel Assay

[Elizabeth Chlipala via Histonet]

Ronnie 

We have these protocols in place for porcine tissue.  We use the roche kit for 
tunel 11684809910 and for CC3 we use the cell signaling antibody 9664.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Houston, Ronald via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, August 16, 2016 9:45 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Caspase 3 and Tunel Assay

Is anyone working on this just now? And more specifically on the pig species?

Any pointers would be appreciated
Thanks

Ronnie Houston, MS HT(ASCP)QIHC FIBMS
Anatomic Pathology Manager
Laboratory Services
700 Children's Drive
Columbus, OH 43205
(P) 614-722-5450
(F) 614-722-2899
ronald.hous...@nationwidechildrens.org
www.NationwideChildrens.org

"Without continual growth and progress, such words as improvement, achievement, 
and success have no meaning."
~ Ben Franklin

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Re: [Histonet] Automated Slide Stainers

[Elizabeth Chlipala via Histonet]

Atoska

I'm pretty sure that the Sakura Tissue Tek Prisma has the capability to hold 2 
x 3 slides with some adaptors  to the slide baskets, I have a fax from 2009 
that list the parts necessary.  The regular 20 slide holders can be changed to 
hold 5 - 2 x 3 slides.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: Atoska Gentry via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Friday, August 05, 2016 9:49 AM
To: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Automated Slide Stainers

Hello, one of my department's researchers is in need of an automated slide 
stainer which accommodates 3"x2" microscope slides. If you have experience with 
such will you please contact me at your earliest convenience? Regards, Atoska 
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Re: [Histonet] PFA Fixed tissue not sticking to slides?

[Elizabeth Chlipala via Histonet]

Are you using a good plus slide?  We have started using these Tru-Bond slides 
when we have tissue adherence problems, they seem to work really well.  How are 
you handling the slides once you section?

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: jenny sandy via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Friday, July 29, 2016 1:48 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] PFA Fixed tissue not sticking to slides?

Dear HistoFolks,

Has anyone had experience with cryo-sectioned tissue that is PFA fixed not 
adhering to slides?

The tissue is rabbit cornea culture, 4% PFA fixed O/N, then run through 20% 
sucrose gradient, then frozen in OCT.

We think that the PFA fix is much longer than needed for a tissue this thin, 
and were going to try 2 hr fix. Would over-fixation cause the section to not 
adhere?  

The PFA/Sucrose fixation really improves the morphology and the IHC stains that 
we have run so far.

Thank you in advance for your help.

Jennifer Eveleth
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Re: [Histonet] block scrapers

[Elizabeth Chlipala via Histonet]

Lauren

I have always used a very dull steak knife (sharp ones are too sharp and will 
cut the cassette) mine is probably over 20 years old (now I'm dating myself).  
I'm old school so I still use that but the rest of the techs will use the para 
trimmer.  The para trimmer is more ergonomically friendly less repetitive than 
a knife -  here is the link to the website

https://www.thermofisher.com/order/catalog/product/B3120205

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

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1567 Skyway Drive, Unit E
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-Original Message-
From: Lauren Sweeney via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, July 21, 2016 12:47 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] block scrapers

Hi all,

My lab is in need of some tools to scrap the paraffin off the edges of the 
blocks after embedding. Does anyone have any recommendations for me?


Thanks!!
L
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Re: [Histonet] Fading of H controls -long response with portions of SOP

[Elizabeth Chlipala via Histonet]

James

We use recycled xylene in our H stainer and have never experienced any fading 
of the eosin.  When you recycle your clearing agent do you check for purity and 
contamination, you have to also take into consideration that xylene substitutes 
may behave differently than xylene.   We have an SOP that governs how we 
recycle, we keep tract of runs and assign lot numbers to recycled products, and 
we also record what lot numbers were used when we change the stainer or tissue 
processer, so if we do have problems (we normally do not) we could trace back 
to the lot of recycled reagent. 

This is how we test for xylene purity - see below.  For our recycled alcohol we 
do merge multiple runs since we will use this to make up solutions but we also 
have a defined procedure for that and it includes determining  the  percentage 
of alcohol via hydrometer and adjusting for temperature prior to making up a 
reagent with it.

Excess alcohol in your xylene can also cause reagents to fade, especially ones 
that are susceptible to fading in alcohol such as some of the red chromogens 
for IHC.  This could be due to the process of recycling or your overall QC and 
how often you change your stainer.

8.6 Testing the Purity of Recycled Xylene or Propar

8.6.1   Pour 85ml of recycled xylene or propar into a clean, dry 100ml 
graduated cylinder, then add 15ml deionized water.
8.6.2   Seal the top of the graduated cylinder and invert the mixture a couple 
of times.
8.6.3   Allow the mixture to settle. Make sure all the water settles to the 
bottom of the graduated cylinder and does not cling to the sides above the 
xylene/ or propar/water separation point (approximately the 15ml level of the 
graduated cylinder).
8.6.4   Record the water/solvent separation point as indicated by the bottom of 
the meniscus on Form 1: Solvent Recycler Log - EQ-0014.1.
8.6.5   Use the following equations to calculate the percent impurities and 
percent purity of the recycled xylene:  
  
(measured separation point) - 14.9 = (% impurities) 
 100 - (% impurities) = (% pure xylene)
8.6.6   Enter the percent pure xylene on Form 1: Solvent Recycler Log - 
EQ-0014.1.

8.7 Assigning Control Numbers for Xylene and Propar

8.7.1   Each recycled lot will be kept in a separate carboy, and assigned a 
unique control number.
8.7.2   Control numbers for xylene have the format: two digit year 
(YY)-RXYL-lot #, and control numbers for propar have the format: two digit year 
(YY)-RPRO-lot #. 
8.7.3   Write the assigned control number and the expiration date (one year 
from recycling) on Form 1: Solvent Recycler Log - EQ-0014.1.
8.7.4   Fill out a reagent control label (see Appendix 1) to label the carboy.
8.7.5   Recycled alcohol will not be assigned control numbers since multiple 
lots are combined and it is only used in making other reagents. Instead, 
control numbers are assigned to the solutions made from recycled alcohol (50%, 
70%, 80%, 95% or other).

8.8 Testing Recycled Alcohol for Xylene Contamination

8.8.1   Every batch of recycled alcohol should be tested for xylene 
contamination.
8.8.2   Mix a small volume of recycled alcohol with an equal volume of 
deionized water. 
8.8.3   If the recycled alcohol is contaminated with xylene, the mixture will 
become cloudy or milky.
8.8.4   If contamination is discovered, dispose of the recycled product, clean 
all containers thoroughly (see 8.1.1), and flush the recycler (see 7.3.7) 
before proceeding with the next batch. 

8.9 Measuring the Percent Alcohol of Recycled Alcohol Using a Hydrometer 

8.9.1   A hydrometer measures specific gravity and is used to determine the 
percent alcohol of recycled alcohol produced by the CBG Biotech Solvent 
Recycler System.
8.9.2   When using recycled alcohol to make alcohol solutions (50%, 70%, 80%, 
95% or other) it is necessary to determine the percent alcohol of the current 
stock of recycled alcohol.
8.9.3   Since water and alcohol have different densities, and therefore 
different specific gravities, the specific gravity of a mixture of these two 
liquids indicates the percent alcohol of the solution.
8.9.4   The temperature of a liquid affects its density, and therefore its 
specific gravity. Because of this, hydrometer reading must be adjusted for 
temperature. 
8.9.5   Since the alcohol collected from the recycler is slightly warm, it is 
best to let it stand at room temperature overnight before using it to make 
alcohol solutions. 
8.9.6   Fill a transparent 500ml graduated cylinder with a sample of the 
recycled alcohol and gently place the hydrometer into the solution, such that 
it is floating with the weighted end down. Allow the hydrometer to stop moving, 
then record the reading (Tralles, not Proof) at the bottom of the meniscus on 
the reagent control form.
8.9.7   Measure the temperature of the alcohol in the graduated cylinder and 
record it on the reagent 

Re: [Histonet] expiration dates

[Elizabeth Chlipala via Histonet]

Linda

We have in our reagent policy that any chemical that does not come with an 
expiration date - we assign it a 5 year from receipt expiration date.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Blazek, Linda via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, July 19, 2016 10:35 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] expiration dates

Does anyone see an expiration date printed on their can of Freeze Spray?
I was told by the company I purchase mine from that it was not needed because 
it was "not a medical device".

Thanks
Linda

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Re: [Histonet] CD34 for primate tissue

[Elizabeth Chlipala via Histonet]

Cathy

Before doing that I would check the sequence homology of the immunogen between 
human and rhesus.  I would do this for all sources of the antibody, if the 
company does not want to provide you with the sequence then technical support 
should be able to blast the sequence for you and get back to you the sequence 
homology, greater than 70% is worth a shot it may not work or it may not.  You 
also need to check if it's the same clone it might be the same antibody just 
distributed by different vendors, check the protein concentration, purification 
method, isotype, and if there are matching images you are likely looking at the 
same antibody so you don't want to waste your time with ordering the same 
antibody but from a different vendor.Remember information on specification 
sheets can be inaccurate - this depends upon the vendor, some vendors actually 
test internally while other vendors rely of user for cross reactivity 
information so you need to take that information with a grain of salt.
   My suggestion is to take a look at the antibody on CiteAb - this website is 
a bit better than biocompare since it rates antibodies by number of citations, 
check to see if the other antibodies have publications with your use case.  The 
link for CiteAb is https://www.citeab.com/ We find with some antibodies that 
work across species that the primary antibody concentration may fluctuate so a 
concentration that works in human may not work in the particular species you 
are working with.  There are many other parameters such as time in fixation 
between your human tissue and rhesus tissue.  Are you getting any hint of 
staining or is it just completely negative.

It's really important to spend some significant time upfront when researching 
antibodies for a new target - you want to biases yourself towards success.  If 
you are an NSH member I posted a worksheet for target development on the BLOCK 
you can access it there.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Cathy M. Mathis/Comparative Medicine via Histonet 
[mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, June 30, 2016 8:07 AM
To: histonet@lists.utsouthwestern.edu
Cc: Eddie Martin
Subject: Re: [Histonet] CD34 for primate tissue

Rhesus is a species of old world monkey.  There are a couple of other companies 
that have this same clone and on their datasheets they say it cross reacts with 
rhesus monkey.  The CD34s (2) that I have do not state on their data sheets 
that they cross react with anything except human and yet are the same clone as 
the companies that say it does.  I guess I will just break down and purchase a 
different clone that states it works in rhesus monkey.
Cathy

-Original Message-
From: Eddie Martin [mailto:edmarti...@gmail.com]
Sent: Wednesday, June 29, 2016 8:22 PM
To: Cathy M. Mathis/Comparative Medicine
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] CD34 for primate tissue

What species is the kidney you are trying to stain with IHC. Because you 
mentioned that you used normal human kidney and it worked , but Rhesus kidney 
didn't stain. I'm wondering if the Rhesus kidney is human or another animal 
species? The Novocastra CD34 (QBEND10) is intended for human tissue. 

Best,
Eddie Martin, HTL,QIHC

Sent from my iPhone

> On Jun 29, 2016, at 4:43 PM, Cathy M. Mathis/Comparative Medicine 
>  wrote:
> 
> 2 more bits of information about my dilemma; I did try staining 
> without any retrieval - no signal I am using rhesus kidney as a 
> control (getting no signal), but I also ran some human kidney and got 
> beautiful staining with a 20 minute HIER using a pH 8 EDTA buffer.
> So I know the antibodies and my protocols work, just not on my rhesus.  Does 
> anyone know of a CD34 that will work in this species?
> More suggestions please?  Thank you all for your time.
> Cathy
> 
> -Original Message-
> From: Eddie Martin [mailto:edmarti...@gmail.com]
> Sent: Wednesday, June 29, 2016 5:34 PM
> To: Cathy M. Mathis/Comparative Medicine
> Cc: histonet@lists.utsouthwestern.edu
> Subject: Re: [Histonet] CD34 for primate tissue
> 
> Hi Cathy,
> 
> EDTA (Retrieval #2) for 20 minutes on the Bond-Rx should be sufficient. And 
> all that is necessary. Please contact me if you need additional help. 
> 
> Best,
> Eddie Martin, HTL, QIHC
> edmarti...@gmail.com
> 
> Sent from my iPhone
> 
>> On Jun 29, 2016, at 8:16 AM, Cathy M. Mathis/Comparative Medicine 
>>  wrote:
>> 
>> Good morning my fellow Histo-netters, Does anyone have experience 
>> with any CD34 antibody that would work in FFPE rhesus tissues?  A few 
>> companies have one with clone QEBend 10 and say that it works but I 

Re: [Histonet] Transport training for formalin fixed specimens

[Elizabeth Chlipala via Histonet]

Gareth

I would think it would have more to do with OSHA than CDC - this could involve 
several modules.  

We have modules that cover the following - this is not all that we train on.  
If you are a small business OSHA can help out.  We have utilized their services 
with a voluntary compliance audit.  We were referred to the Chemical Hygiene 
Officer at the local university and they helped us out.

*   Formaldehyde Awareness
*   General Safety Awareness 
*   Hazard Communication
*   Laboratory & Chemical Hygiene

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: Gareth Davis via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, June 29, 2016 5:18 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Transport training for formalin fixed specimens

Okay, is there a training course for just transportion formalin fixed 
specimens.  CAP told me last year that anyone picking up specimens for us has 
to go through a training course.  The inspector seemed to be more concerned 
with the employees knowledge of formalin and possible spills.
So, does anyone know what course I am suppose to be using?
 I have looked on the CDC website, but all the courses I find refer to specific 
infectious disease.
Thanks in advance.
--
Gareth B. Davis, HT (ASCPcm), QIHC
Yuma Gastroenterology
Yuma, AZ 85364
928-248-5259
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Re: [Histonet] using FFPE positive control for frozen tissue IHC?

[Elizabeth Chlipala via Histonet]

There are many companies that sell frozen tissue both normal and diseased.  
Frozen IHC does not normally require retrieval most FFPE IHC does.  I don't 
think it would be good practice to do what you are suggesting.

Liz 

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Preiszner, Johanna via Histonet 
[mailto:histonet@lists.utsouthwestern.edu] 
Sent: Monday, June 27, 2016 8:48 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] using FFPE positive control for frozen tissue IHC?

Hi,

We have difficulty obtaining frozen tissue to use as positive control when we 
do IHC on frozen sections...

Is it allowed to use FFPE sections for this purpose? If not, can someone 
explain to me why not? Is it wrong by principle or prohibited by regulations? 
If it's the regulations, can someone give me a reference, please?

I have not seen an antibody that works on FFPE tissue and refuses to work on 
frozen tissue...And the manufacturers always provide info about the type of 
tissue the antibody would work with.

Thank you,
Hanna Preiszner
ETSU/QCOM
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Re: [Histonet] CD3 antibody for FFPE mouse tissue

[Elizabeth Chlipala via Histonet]

Brett

Dako's rabbit anti human CD3 cross reacts with mouse, or where you looking for 
a CD3 that only detects mouse?

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 881-0763 cell
(303) 682-9060 fax
l...@premierlab.com

Ship to address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

From: Connolly, Brett M via Histonet [histonet@lists.utsouthwestern.edu]
Sent: Tuesday, June 21, 2016 7:59 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] CD3 antibody for FFPE mouse tissue

Hi all,

Looking for recommendation for an anti-mouse CD3 antibody for FFPE sections.

I have some old Histonet messages referring to a Neomarkers RB-360 ab which I 
found through Thermo, but I would like to hear about more recent 
experiences/recommendations

Thanks as always,
Brett

Brett M. Connolly, Ph.D.
Prin. Scientist,
Translational Biomarkers - Imaging
Merck & Co., Inc.
PO Box 4, WP-44K
West Point, PA 19486
brett_conno...@merck.com
T- 215-652-2501
F- 215-993-6803

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Re: [Histonet] Sakura Prisma 4740 combo film

[Elizabeth Chlipala via Histonet]

In our situation the charcoal filters were not sufficient enough, we used 
xylene and it was not the tape but glass coverslipper.  We tried it with just 
the carbon filters and it was not good enough.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Jonathan Jennings [mailto:jjenni...@thedermlab.com] 
Sent: Friday, June 03, 2016 7:57 AM
To: Elizabeth Chlipala
Subject: RE: [Histonet] Sakura Prisma 4740 combo film

Ours has 2 charcoal filters located behind the carousel rack on top of the 
coverslipper.  I believe the filters have to be changed once per year.  This 
would eliminate having to have external exhaust.  We don't have any issues with 
fumes from our unit, but we also are using xylene substitute (SubX) inside the 
stainer, except for the last station before the slides go into the 
coverslipper.  We use xylene in the last station of the stain line and xylene 
inside the coverslipper.  Of course the only reason we are using xylene in 
these 2 stations is to activate the hydrocarbons in the tape coverslip film, as 
xylene substitute will not work for this.

Jonathan Jennings
Lab Manager
205.705.3550
205.705.3554
jjenni...@thedermlab.com



3918 Montclair Rd, Ste 105
Birmingham, AL 35213
www.thedermlab.com 

This e-mail transmission, and any documents, files or previous e-mail messages 
attached to it, may contain confidential information. If you are not the 
intended recipient, or a person responsible for delivering it to the intended 
recipient, you are hereby notified that any disclosure, copying, distribution 
or use of any of the information contained in or attached to this message is 
strictly prohibited. If you have received this transmission in error, please 
immediately notify Jonathan Jennings by reply email or by telephone 
1-855-705-1776, and destroy the original transmission and its attachments 
without reading them or saving them to any media storage device. 
 


-Original Message-
From: Elizabeth Chlipala via Histonet 
[mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, May 31, 2016 9:50 AM
To: Patti Nelson - PNP Lab Consultant <nelsonr...@verizon.net>; 
'histonet@lists.utsouthwestern.edu' (histonet@lists.utsouthwestern.edu) 
<histonet@lists.utsouthwestern.edu>
Subject: Re: [Histonet] Sakura Prisma 4740 combo film

Patti

We have the Sakura Prisma with the Glas coverslipper and we have ours vented 
out.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Premier Laboratory, LLC PO Box 18592 
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Patti Nelson - PNP Lab Consultant via Histonet 
[mailto:histonet@lists.utsouthwestern.edu]
Sent: Tuesday, May 31, 2016 7:28 AM
To: Histonet@lists.utsouthwestern.edu
Subject: [Histonet] Sakura Prisma 4740 combo film


Hi everyone,

I am considering placing a Sakura Prisma 4740 combo film in one of my labs. My 
question is , does the cover slip unit need an external exhaust. Please 
consider that the space the unit will be placed is small. Thank you in advance.



Sincerely,

PATTI NELSON  H.T.(ASCP) 
PNP LABORATORY CONSULTANTS
SUPERVISOR DGC/ZADEH LABS
PO BOX 412
CABAZON, CA. 92230
909-841-9761
nelsonr...@verizon.net
CONFIDENTIALITY NOTICE:This message and any included attachments are from Patti 
Nelson, PNP Laboratory Consultants and are intended only for the addressee. The 
information contained in this message is confidential and may contain 
privileged, confidential, proprietary and/or exemption from disclosure under 
applicable law.  Unauthorized forwarding, printing, copying, distribution, or 
use of such information is strictly prohibited and may be unlawful.  If you are 
not the addressee, please promptly delete this message and notify the sender 
ofthe delivery error by e-mail or you may call  909-841-9761.

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Re: [Histonet] Sakura Prisma 4740 combo film

[Elizabeth Chlipala via Histonet]

Patti

We have the Sakura Prisma with the Glas coverslipper and we have ours vented 
out.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Patti Nelson - PNP Lab Consultant via Histonet 
[mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, May 31, 2016 7:28 AM
To: Histonet@lists.utsouthwestern.edu
Subject: [Histonet] Sakura Prisma 4740 combo film


Hi everyone,

I am considering placing a Sakura Prisma 4740 combo film in one of my labs. My 
question is , does the cover slip unit need an external exhaust. Please 
consider that the space the unit will be placed is small. Thank you in advance.



Sincerely,

PATTI NELSON  H.T.(ASCP) 
PNP LABORATORY CONSULTANTS
SUPERVISOR DGC/ZADEH LABS
PO BOX 412
CABAZON, CA. 92230
909-841-9761
nelsonr...@verizon.net
CONFIDENTIALITY NOTICE:This message and any included attachments are from Patti 
Nelson, PNP Laboratory Consultants and are intended only for the addressee. The 
information contained in this message is confidential and may contain 
privileged, confidential, proprietary and/or exemption from disclosure under 
applicable law.  Unauthorized forwarding, printing, copying, distribution, or 
use of such information is strictly prohibited and may be unlawful.  If you are 
not the addressee, please promptly delete this message and notify the sender 
ofthe delivery error by e-mail or you may call  909-841-9761.

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Re: [Histonet] thank you HistoNet! Histology STEM long off-topic

[Elizabeth Chlipala via Histonet]

The NSH as a part of its awards and scholarships program has the Newcomer 
Helping Hand Scholarship.  

If you are considering doing something like what Adelle or Ray did  and are a 
NSH member for two years you can apply for this scholarship.  The scholarship 
is for $2000.00 so it is quite substantial.  The deadline for awards nomination 
is June 1st, so there is still time to nominate yourself.

Here is the link  http://nsh.org/content/newcomer-helping-hand-scholarship

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Ray via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Friday, May 27, 2016 9:49 AM
To: Adelle Schade
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] thank you HistoNet! Histology STEM long off-topic

Histonet- 
  
I cringe at the thought of being dinged for taking time with something somewhat 
tangential to histology but here I go.  If histology as part of STEM (science, 
technology, engineering, math) and kids' futures is a bit overboard for you, 
please use your delete button now. 
  
First Adelle Schade should be congratulated profusely for her work with her 
students and starting their high school histology/biomedical research lab.  I 
myself helped and donated to a local (Seattle) high school biotech/biomed lab 
where we (they) did some fairly sophisticated IHC and molecular techniques. I 
am also very familiar with the Intel International Science and Engineering 
Competition in Phoenix.  We in Washington state have recently sent 20 kids 
there and they have returned.  Involved with STEM educational outreach for our 
state K-12 for 15 years, I am currently re-elected to our Board of the state 
fair, WSSEF (Washington State Science and Engineering Fair).  Intel affiliated, 
we sent those 20 or so kids to Phoenix, along with Adelle's kids, to compete 
against close to 2,000 high schoolers from every state and 70 countries around 
the world. 
  
Our son is safely working on his Masters degree in Aeronautical Engineering so 
he will be in good shape.  But I am very worried for many, many kids in the US 
for an almost systemic rejection of science in lieu of nearly 100% sports, art 
and music.  I love sports having played baseball in high school and college.  I 
love artistic endeavors be it at a symphony, art museum or stage play.  I love 
music even though it might be quite different from the music many enjoy today.  
But I also love to eat well and live well and enjoyed saving up for a 
comfortable retirement.  Can you do those things in non-STEM pursuits? Of 
course you can!  Yet the fact is that very, very, very few K-12 students will 
ever make a great future life as a professional athlete, artist or musician.  
Yet in the world of the future, the majority of great jobs are in STEM.  That 
is not an opinion.  That is simply reality. 
  
So I encourage anyone to get involved with teachers in high school such as 
Adelle Schade. Histology is obviously the topic of discussion here but it can 
be with anything STEM.  Depending on which study you look at, we (the US) are 
15th-25th amongst nations in the world in K-12 science and math education.  Far 
too many of the kids in the US are being left behind for the jobs of the 
future. 
  
Having "retired" to Spokane Washington, I now find myself as a part-time 
lecturer at the University of Washington Medical School-Spokane campus, 
lecturing in microanatomy to first year medical students in their pre-clinical 
required curriculum.  Yet I still will be helping K-12 students around here in 
their local science fairs and also with the Washington State Science and 
Engineering Fair in preparation for the May 2017 INTEL international fair to be 
held in Los Angeles, CA.  A few more histology/molecular/IHC/biotech/biomed  
projects would be AWESOME. 
  
Thanks to people like Adelle and I encourage everyone with helping K-12 kids 
and their teachers with educational outreach for science fairs in histology or 
anything STEM . 
  
Ray Koelling 
UW Med School-Spokane campusl lecturer, microanatomy 
- Original Message -

From: "Adelle via Histonet Schade"  
To: "histonet@lists.utsouthwestern.edu"  
Sent: Friday, May 27, 2016 4:29:53 AM 
Subject: [Histonet] thank you HistoNet! 

Hello Histonet, 
I am a high school science teacher and part-time PhD student in Cell and 
Molecular Biology. I started a histology/ biomedical research laboratory in my 
high school two years ago and it has been quite an experience.  This year, two 
of my students who incorporated histological testing into their science fair 
project protocols won awards at the INTEL International Science and Engineering 

Re: [Histonet] shrinking mouse eyes

[Elizabeth Chlipala via Histonet]

Nancy

I cannot comment on the plastic embedding but this also happens with paraffin 
embedded samples.  We trim off one of the edges on all mouse and rat eye 
samples and that eliminates that problem with paraffin embedded samples.  I can 
send pictures of a rat eye that has been trimmed.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Thomas, Nancy via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, May 18, 2016 1:30 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] shrinking mouse eyes

Dear experts,
We often get a request to embed and section mouse eyes in GMA.  Currently we 
have been working with samples from 1 month old mice.  The big problem we need 
to solve is that the shape of the eyes is fine up until we embed them.  I'm 
sure the heat from the exothermic reaction is causing the eyes to sink inward, 
losing their round shape, but  what can prevent this?   We use the Immuno-bed 
kit which sets up fine and sections well.  Is there something more I can do 
during dehydration and infiltration steps?  We have experimented with longer 
dehydration times, but haven't seen much difference.  The eyes were fixed in 
Davidson's for 24 hr.  Would longer fixation help?
Thank you in advance for all advice,

Nancy Thomas
Senior Lab Manager, Histology Core
Stowers Institute for Medical Research
1000 E. 50th Street
Kansas City, MO  64110

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Re: [Histonet] Cryostat Chirp

[Elizabeth Chlipala via Histonet]

Gary

I think that might have to do with the fan motor inside the unit, it might need 
to be replaced.   We had ours replaced a few years ago.  Do you service/PM the 
cryostat yearly?

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Martin, Gary via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, May 03, 2016 2:46 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Cryostat Chirp

My Leica CM 1850 cryostat is making a chirping sound and I can't figure out 
what is doing this. Has anyone experienced this sound, if what was the resolve. 

Thanks 

Gary

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Re: [Histonet] alcohol or formalin filtration system

[Elizabeth Chlipala via Histonet]

We have used the filtration systems in the past - the one thing you need to 
keep in mind is that the percentage of alcohol you put in is the percentage you 
get out.  You need to test with a hydrometer and account for temperature what 
you get out.So you only want to put in your absolute and possibly your 95% 
if you put in 70% or 80% at the same time you will decrease your concentration 
too much.  

We use a fractional distillation unit now and even with that we get out ranges 
from 90 to 95% for alcohol when we account for temperature when using a 
hydrometer.  We are also able to recycle our xylene, and propar with the same 
unit so it has helped us out a lot with both the amount of waste we generate 
and especially the amount of xylene that we need to order.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Baldwin, Kathy via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, April 20, 2016 9:55 AM
To: 'histonet@lists.utsouthwestern.edu'
Subject: [Histonet] alcohol or formalin filtration system

Hi Histonetters
Is anyone currently using the alcohol or formalin filtration systems and if you 
are can I have your thoughts??


Thanks
S. Kathy Baldwin
Histology/Cytology Supervisor
Memorial Hospital and Health Care Center
800 West 9th St.
Jasper, Indiana  47546
Office 812-996-0210
Fax 812-996-0232





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Re: [Histonet] Validating IHC on decalcified specimens

[Elizabeth Chlipala via Histonet]

Teri

We have a spot on all of our IHC SOP's that will state if the antibody has been 
tested on decalcified samples.   We do not routinely add decalcified tissue to 
our routine IHC protocol development runs, so for example if the target is a 
soft tissue target we will develop on a variety of soft tissues that express 
different  staining intensities.  If the target is expressed in bone and 
decalcified bone is the target tissue we will work with both decalcified and 
soft tissue during the development process.If we are taking a previously 
soft tissue antibody and testing it in bone or a sample that has been 
decalcified then that information is added to our internal tracking documents 
and then the SOP as a revision, we follow the same protocol if we are testing 
on another species that we have not previously stained for.  

I can give you an example of the last scenario.  We are frequently requested to 
run GFAP IHC sometimes the samples are routinely fixed on other occasions the 
skull has been left intact and the sample decaled, we initially developed and 
verified on soft tissue but later added to the verification that the antibody 
was tested on decaled samples and we would list the decalcification method/s 
tested in the SOP.  I'll send off examples of both our GLP and non-GLP IHC 
protocols in a different e-mail.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: Teri Johnson via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, April 05, 2016 12:50 PM
To: 'histonet@lists.utsouthwestern.edu'
Subject: [Histonet] Validating IHC on decalcified specimens

Hi Histonetters,

Can someone tell me if they do anything specific to validate their IHC markers 
on decalcified specimens? If no, do you test them anyway?

Teri Johnson
Manager, Clinical Trial Testing
Genoptix, Inc., a Novartis company
BioPharma
1811 Aston Avenue
Carlsbad, CA  92008
USA

Phone +1 760 516 5954
tejohn...@genoptix.com
www.genoptix.com




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confidential and proprietary to Genoptix Medical Laboratory or its 
subsidiaries. Any unauthorized review, use, disclosure or distribution is 
prohibited. If you are not the intended recipient, immediately contact the 
sender by e-mail and destroy all copies of the original message.
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Re: [Histonet] Porcessing FFPE tissue without alcohol??

[Elizabeth Chlipala via Histonet]

Carl

We have tried multiple procedures, there are procedures that use a combination 
of 2% osmium and 5% postassium dichromate, ones that call for shorter periods 
of time in Osmium and then periodic acid rinse post osmium - this is referenced 
in Freida's book - Histotechnology a Self-Instructional Text.  We ultimately 
settled on a modification of different methods.  We use a 1% osmium solution 
for 24 hours rinse well in water and process same day short cycle (20 minutes 
per station with propar instead of xylene).  I'll post some images and our 
procedure on the block.  Sectioning and staining of these samples is tricky and 
we have found at least in our hands that the tissue is very friable (liver 
primarily, sciatic nerve and muscle samples turned out better) and does not 
stay on the slides well so we have not been able to even counterstain with H 
without considerable tissue loss.   Sections can be a bit uneven which is OK 
for routine viewing via a microscope but not good for whole slide scanning and 
image analysis applications.  I have pics of all of the problems associated 
with this protocol along with some really nice ones.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: Hobbs, Carl [mailto:carl.ho...@kcl.ac.uk] 
Sent: Saturday, March 26, 2016 12:56 PM
To: Elizabeth Chlipala; Joanna; Rene J Buesa
Cc: histonet
Subject: Re: [Histonet] Porcessing FFPE tissue without alcohol??

Thanks, Liz.
If you look at fat all the time, using Osmium.you then are not sure if you 
use K-dichromate?
I am a tad confused

Alsowhy not trim the block too much?

Best wishes,

Carl
NB: Rene stated that I wouldn't be able to use any other fat stains...that's 
the point, Rene.
I don't need any other.
I commit to Osmium.

Yep...there are many variations for fat staining.
Imho...most are histochemical mythology.
Osmium "stains" fat...histologically.
As do  the conventional fat-soluble dyes when using frozen sections.
I would only listen to alternatives/disagreements from JKiernan.

I am still waiting for the next "generation" Histo person.
Cook, Kiernan.who are the other Seminals??

Enquiringly

Carl

________
From: Elizabeth Chlipala <l...@premierlab.com>
Sent: 26 March 2016 15:58
To: Joanna; Rene J Buesa
Cc: Hobbs, Carl; histonet
Subject: RE: [Histonet] Porcessing FFPE tissue without alcohol??

We use osmium post fixation to look at fat all of the time in mouse liver, 
nerve and muscle samples.  It works well, sample size needs to be thin, samples 
are friable and can crack easily.  We use a specific procedure for this it 
includes potassium dichromate I think, I'm at home but on Monday I can send the 
reference.  One more thing don't trim into the block too much.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Premier Laboratory, LLC PO Box 18592 
Boulder, CO 80308
(303) 682-3949 office
(303) 881-0763 cell
(303) 682-9060 fax
l...@premierlab.com

Ship to address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

From: Joanna via Histonet [histonet@lists.utsouthwestern.edu]
Sent: Saturday, March 26, 2016 9:20 AM
To: Rene J Buesa
Cc: Hobbs, Carl; histonet
Subject: Re: [Histonet] Porcessing FFPE tissue without alcohol??

How about Sudan Black stain?

> On Mar 26, 2016, at 4:32 AM, Rene J Buesa via Histonet 
> <histonet@lists.utsouthwestern.edu> wrote:
>
> The only problem I see is that the fat will be preserved, as you 
> wrote, as a black osmium oxidate but you will not be able to use any 
> "standard" fat stain; otherwise it will work.René
>
>On Friday, March 25, 2016 2:41 PM, "Hobbs, Carl via Histonet" 
> <histonet@lists.utsouthwestern.edu> wrote:
>
>
>
>
> Fix the tissue in Formalin, wash well in dw, then place very small 
> pieces into Osmium tetroxide solution ( std soln for TEM post-fixation) 
> Processing to Pwax as usual.
> Basically, you will see lipids as black ( oxidised osmium) That's the 
> only way to demonstrate solvent- soluble lipids, using Pwax processing.
> Sure, there are caveats but, in the main...it will be Ok, imho.
> I invite comments as I may be doing exactly this very soon, to count 
> myelinated nerve fibres in a sciatic nerve.
>
>
>
>
>
> Carl Hobbs FIBMS
> Histology and Imaging Manager
> Wolfson CARD
> Guys Campus, London Bridge
> Kings College London
> London
> SE1 1UL
>
> 020 7848 6813
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> http://lists.utsouthwestern.edu/mailma

Re: [Histonet] Porcessing FFPE tissue without alcohol??

[Elizabeth Chlipala via Histonet]

We use osmium post fixation to look at fat all of the time in mouse liver, 
nerve and muscle samples.  It works well, sample size needs to be thin, samples 
are friable and can crack easily.  We use a specific procedure for this it 
includes potassium dichromate I think, I'm at home but on Monday I can send the 
reference.  One more thing don't trim into the block too much.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 881-0763 cell
(303) 682-9060 fax
l...@premierlab.com

Ship to address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

From: Joanna via Histonet [histonet@lists.utsouthwestern.edu]
Sent: Saturday, March 26, 2016 9:20 AM
To: Rene J Buesa
Cc: Hobbs, Carl; histonet
Subject: Re: [Histonet] Porcessing FFPE tissue without alcohol??

How about Sudan Black stain?

> On Mar 26, 2016, at 4:32 AM, Rene J Buesa via Histonet 
>  wrote:
>
> The only problem I see is that the fat will be preserved, as you wrote, as a 
> black osmium oxidate but you will not be able to use any "standard" fat 
> stain; otherwise it will work.René
>
>On Friday, March 25, 2016 2:41 PM, "Hobbs, Carl via Histonet" 
>  wrote:
>
>
>
>
> Fix the tissue in Formalin, wash well in dw, then place very small pieces 
> into Osmium tetroxide solution ( std soln for TEM post-fixation)
> Processing to Pwax as usual.
> Basically, you will see lipids as black ( oxidised osmium)
> That's the only way to demonstrate solvent- soluble lipids, using Pwax 
> processing.
> Sure, there are caveats but, in the main...it will be Ok, imho.
> I invite comments as I may be doing exactly this very soon, to count 
> myelinated nerve fibres in a sciatic nerve.
>
>
>
>
>
> Carl Hobbs FIBMS
> Histology and Imaging Manager
> Wolfson CARD
> Guys Campus, London Bridge
> Kings College London
> London
> SE1 1UL
>
> 020 7848 6813
> ___
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
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Re: [Histonet] Image analysis software

[Elizabeth Chlipala via Histonet]

Judi

Through the years we have used a wide variety of software applications, we have 
been performing image analysis since 2006, we started whole slide scanning in 
2007.  We have used various modules from Zeiss, Image Pro, Aperio's Toolbox, 
Definiens and free shareware such at Image J.  There are also new vendors on 
the market such as Indica Labs and Visopharm.  I think you need to make an 
educated decision based upon what your needs are and how complex your analysis 
is and the price of the product, some of the commercially available software 
packages can be expensive.  I saw the earlier post that discussed Image J and 
that might be a good option to start with if you have the time and ability to 
learn how to work with the software, you would be surprised to learn that Image 
J can do quite a bit.  The only thing that it may not be capable of is pattern 
recognition, others that use the software can comment on that aspect.  I do 
know that it is easier to trace in Image J than it is in some of
  the viewer software that are linked to a particular scanner. 

The other software solutions may be a bit easier to understand and work with 
initially but there will always be a learning curve associated with any 
software package that you choose to use.  Most software packages will support 
different file types, such as tiff or modified tiff, you will need to make sure 
the image format your scanner generates is compatible with the software you 
choose.   My suggestion would be to look at all of them and determine what is 
best for your use cases.  I would also be a bit forward thinking, you might 
think that you only need the software to do certain things but as you use the 
technology more you will see that you will want to use if for other use cases.  

The other thing I want to comment on is that the success of your image analysis 
is not just related to the software that you choose to use  but also the 
quality, consistency and reproducibility of your histology preparations.  

If you want more information on the topic I would suggest going to the DPA - 
Digital Pathology Association website, it has a list of most of the vendors in 
the space and if you become of member, its $50.00 for technologists, you will 
have access to all of the past presentations from the Pathology Visions 
conferences and webinars.  Here is the link to the website.  The other option 
is to attend the Pathology Visions conference, it a conference geared towards 
digital pathology.  If you are a NSH member there is a scholarship available to 
attend the conference, you could apply for that.  The links to the NSH 
scholarship and the Pathology Visions conference are below. The list of vendors 
is under the resources tab.  

https://digitalpathologyassociation.org/

https://digitalpathologyassociation.org/pathology-visions-2016

http://nsh.org/content/digital-pathology-association-scholarship-pathology-vision

Good Luck

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: Judi Ford via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, February 25, 2016 5:36 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Image analysis software

Hi everyone,
We are in the middle of looking at image analysis software and I was wondering 
what software programs people are using. Why did you pick the one you are 
using? Are you using it for brightfield whole slide analysis or for fluorescent 
slide analysis?  What file types can be used with your software?

I would love to hear your experiences.

Thanks,
Judi
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Re: [Histonet] CAP

[Elizabeth Chlipala via Histonet]

Bernice

We are not a clinical lab, we are a GLP compliant lab and we have a procedure 
that addresses this and everything else about reagent preparation.  I will put 
the procedure and forms that we use on the NSH BLOCK for anyone who is 
interested.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Bernice Frederick via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Friday, February 26, 2016 6:57 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] CAP

ANP21382. What kind of policy or SOP do you all have for this question? T asks 
how reagents are given an expiration. This includes but is not restricted to 
reagents where manufacturer does not specify a date. We date made up reagents 
as a 6 month expiration unless we know it doesn't last that long. Came up 
during interim self inspection.
Thanks,
Bernice

Bernice Frederick HTL (ASCP)
Senior Research Tech
Pathology Core Facility
Robert. H. Lurie Cancer Center
Northwestern University
710 N Fairbanks Court
Olson 8-421
Chicago,IL 60611
312-503-3723
b-freder...@northwestern.edu

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Re: [Histonet] Human nuclear antibody MAB1281

[Elizabeth Chlipala via Histonet]

Eva

We have worked with various antibodies in order to detect human cells in a 
rodent background.  I know that this antibody has been referenced in the 
literature and I also have known of other individuals who have had success with 
it, but in our hands we have never been able to make it work on FFPE tissues 
but I believe it works in frozen sections.  We have opted to try some different 
antibodies. 

NuMA -we researched this protein and determined that it may work -here is a 
snippet of what we found from a quick look into the Human Protein Atlas entry 
for NUMA1. Though it is functionally involved in the structural rearrangement 
of DNA during mitosis and apoptosis, it is a constitutively expressed 
structural protein. From the images on HPA 
(http://www.proteinatlas.org/ENSG0137497-NUMA1/tissue/primary+data)  it 
looks like it is almost universally expressed, with the one exception of 
hepatocytes. The vast majority of neurons, which are overwhelmingly 
post-mitotic, show fairly strong expression, so that's encouraging.   We 
initially looked at the cell signaling antibody (our target was human cells in 
a rat background) but found that it does cross react with rat, its fine with 
mouse but this antibody is our hands was found to be sensitive to time in 
fixation and retrieval time and temp.  We opted to try another source for this 
antibody - abcam.   They had several different antibod
 ies for that target.  We work with their antibodies a lot and they normally do 
not list the sequence of the antigen so we could not check the sequence 
homology to other species ourselves but you can call them and they will provide 
that information.  Here is what they provided to us -   "We have not tested 
ab86129 for cross reactivity with rat and we have not received any researcher 
feedback for using the antibody with this species. The immunogen sequence has 
56% identity with the rat protein, so it is unlikely that it will cross react. 
However we have not confirmed this experimentally and we do not know if it 
would show non-specific staining with rat. I do think ab86129 would be the best 
antibody to try since it has the lowest homology with the rat protein. The 
other antibodies that have not been tested with rat have 69% (ab55767) or 88% 
(ab84680) homology."  Ab86129 worked very nicely in our hands.

HLA-1 this will also work nicely but the protein is expressed in the cytoplasm 
and expression levels can vary but it does work nicely in some instances.

So for us its dependent upon the project as to which antibody we will use but 
the NuMA is expressed in the nuclei so it will work quite nicely for dual 
staining of another protein that is expressed in the cytoplasm or cell membrane.

Good Luck

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Eva Permaul via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, February 25, 2016 7:07 AM
To: histonet
Subject: [Histonet] Human nuclear antibody MAB1281

Good morning,
We recently bought MAB1281 with the hope of being able to determine if the 
cells in a mouse model was human or mouse.
We want to use it to stain FFPE tissues. I have tried it with Citrate per the 
companies recommendation. I ran it on a human tonsil but saw no staining at all.
Is there anyone who has been able to get this antibody to work? And if so would 
you please share your protocol?
Thank you,
Eva
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[Histonet] FW: Scholarship Offered by Digital Pathology Association

[Elizabeth Chlipala via Histonet]

FYI – See below  and while I have your attention there are lots of other Awards 
and Scholarships that NSH offers its members.  So nominate yourself or one of 
your colleagues.  It easy to do, all the information is at the link below.

http://www.nsh.org/content/nsh-awards-and-scholarships

Liz
NSH Awards Chair

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com<mailto:l...@premierlab.com>
www.premierlab.com<http://www.premierlab.com/>

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

From: Digital Pathology Association 
[mailto:digital_pathology_associat...@mail.vresp.com]
Sent: Wednesday, February 24, 2016 12:03 PM
To: Elizabeth Chlipala
Subject: Scholarship Offered by Digital Pathology Association

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Scholarship Offered by Digital Pathology Association



The DPA, offers a scholarship intended to support the advancement of knowledge 
and continuing education in digital pathology. It is presented to a National 
Society for Histotechnology (NSH) member certified in histology, and currently 
utilizing Whole Slide Imaging (WSI) in the clinical or research laboratory.

The amount of the scholarship is $1,500 and is to be used for the purpose of 
attending Pathology Visions, the annual meeting of the DPA. Pathology Visions 
provides attendees the opportunity to learn about real-world, practical 
applications in the ever-evolving field of digital pathology through workshops 
and presentations which are separated into three tracks – Education/Research, 
Clinical, and Image Analysis.

Attendees at Pathology Visions get direct access to industry leaders and the 
chance to see the latest product solutions. Pathology Visions 
2016<http://cts.vresp.com/c/?DigitalPathologyAsso/39d05c4973/005a3a31bd/1e74d8ed58>
 is taking place October 23 - 25 in San Diego, CA.

All applications must be completed and submitted to NSH by June 1, 2016. To 
learn more about the scholarship and apply, click 
here<http://cts.vresp.com/c/?DigitalPathologyAsso/39d05c4973/005a3a31bd/89a0f280c9>.
 The recipient of the scholarship will be selected by NSH.

About the Digital Pathology Association
The Digital Pathology Association, located in Indianapolis, IN, was founded in 
2009. Its mission is to facilitate education and awareness of digital pathology 
applications in healthcare and life sciences. Members are encouraged to share 
best practices and promote the use of technology among colleagues in order to 
demonstrate efficiencies, awareness, and its ultimate benefits to patient care. 
To learn more about the DPA and its members and membership opportunities, 
please visit 
https://digitalpathologyassociation.org<http://cts.vresp.com/c/?DigitalPathologyAsso/39d05c4973/005a3a31bd/05f8865950>.



10293 N. Meridian Street | Suite 175 | Indianapolis, IN 46290 | P 317.816.1630 
| F 317.816.1633 | E 
i...@digitalpathologyassociation.org<mailto:i...@digitalpathologyassociation.org>



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Re: [Histonet] NSH Continue education award

[Elizabeth Chlipala via Histonet]

Lin

The link to nominate one of your students for one of the NSH Student 
Scholarship is below.  Deadline for nomination is March 1, 2016.  We have tried 
to make the process easier this year for the program directors/education 
coordinators the application process consists of two fillable forms that can be 
downloaded from the NSH website.   There are a few requirements for these 
awards the individual must be a student member of NSH and they need to be 
enrolled in a formal program.

http://www.nsh.org/content/student-scholarships-1

If you have an individual who is not enrolled in a formal program then my 
suggestion would be to nominate them for one of the professional scholarships 
that NSH has to offer.  In order to be eligible for these scholarships they 
need to be an NSH member.  The deadline for nomination for these scholarships 
is June 1, 2016.  The link is below.

http://www.nsh.org/content/professional-scholarships

So while I have everyone's attention I will be providing a shameless plug for 
the rest of the NSH Awards and Scholarships.  I'm sure you have heard me say 
over and over again how great it is that NSH can provide so many awards and 
scholarships to its members.  It's not really that hard to nominate yourself or 
one of you colleagues for an award, the NSH office and the Awards Committee has 
worked on trying to simplify the process.  Please take the time to nominate an 
individual or a lab that you think has raised the bar for one of the many 
awards and scholarships.  If you need some help please e-mail me and I would be 
more than willing to work with you through the process.

Thanks

Liz - NSH Awards Chair

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: Bustamante, Lin via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, February 10, 2016 10:09 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] NSH Continue education award

I would like to nominate a student I have. Can someone guide me what to do next?
Thank you very much.
Lin.

Lin S. Bustamante B.Sc. H.T.(ASCP)
Research Associate
Texas A University
College of Veterinay Medicine
VIBS Histology Laboratory Supervisor
Room 107 VMA
College Station, Texas 77843-4458
(979)845-3177
(979)458-3499 Fax

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[Histonet] Letters of Recommendation for NSH Awards

[Elizabeth Chlipala via Histonet]

Hello Everyone

I have decided to post this since I have received some e-mails back regarding 
the awards process.  I have listed below my thoughts on Letters of 
Recommendation.

Tips to Writing an Excellent Letter of Recommendation

The Awards Committee has to sift through multiple nominations in order to 
choose the appropriate person for a particular award or scholarship.  We 
receive the persons CV (if they choose to submit, it's not required for some of 
the awards/scholarships, but I would suggest that if you have one you should 
submit it with the application) some info on the nominee,  but what really 
makes one individual stand out from the rest?  It's their letters of 
recommendations, so here are a few tips:


1.   Get focused - understand what award or scholarship you are nominating 
them for and focus in on the description and criteria listed on the NSH website.



2.   Get personal - what has that person accomplished?  Don't generalize, 
we don't just want to know that they are a great histotech, we know that, 
that's why they have been nominated we want to hear WHAT makes them a great 
histotech, tell us what they have done, give us some examples, what makes this 
person stand out amongst others, the more personal the better.



3.   Get multiple people to write letters - everyone has different 
experiences and different viewpoints.  Bottom line the more information the 
better.



4.   And last of all - Get busy and nominate!!!  The deadline for 
nominations is June 1st. http://www.nsh.org/content/nsh-awards-and-scholarships
Thanks

Liz - NSH Awards Chair

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

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Re: [Histonet] Organizing IHC

[Elizabeth Chlipala via Histonet]

Lesley

We have a folder for each antibody, this folder contains all of the information 
on that antibody.  It includes all of our protocol development information, the 
slides from protocol development in the flat plastic holders, spec sheets, 
internet searches, publications and references.  All of the documentation and a 
copy of the current SOP.  If you do not want to have a physical folder this can 
all be done digitally for the paperwork and then if you have a scanner the 
slides from protocol development and validation are scanned, and then 
everything is digital.  We scan all of our protocol development and validation 
slides for IHC since sometimes we may have to return those to the client.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Lesley Bechtold via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, February 04, 2016 11:31 AM
To: 'histonet@lists.utsouthwestern.edu'
Subject: [Histonet] Organizing IHC

Good Afternoon,

I have a question and I'm hoping for some creative answers.  How do other labs 
track their IHC?  Right now, we have 3 large binders with the paper work-up 
sheets and package information filed alphabetically.  We have nothing 
electronic other than an Excel spreadsheet with optimized antibodies listed on 
it.  I'm wondering if there is something out there that other people use rather 
than paper sheets correlated to an Excel file.

Thank you.

Lesley

Lesley S. Bechtold
Senior Manager, Histopathology Sciences
The Jackson Laboratory
600 Main St.
Bar Harbor ME 04609
207-288-6322


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Re: [Histonet] clearing time

[Elizabeth Chlipala via Histonet]

Mohamed

For larger samples we have ranges from 1.5  hours to 6 hours a station.  
Anytime we have larger samples we always utilize three absolute alcohols and 
three xylenes, for your samples I would try 1.5 to 2 hours per station to start.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: mohamed abd el razik via Histonet 
[mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, February 04, 2016 11:52 AM
To: Histonet@lists.utsouthwestern.edu
Subject: [Histonet] clearing time

Dear all 
what is your optimal clearing time in xelene for prostate and tests of dog 
samples (about 0.5 cm) ??is there a need for additional clearing in methyl 
benzoat ?


thanks 
Mohamed

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Re: [Histonet] best glass coverslipper

[Elizabeth Chlipala via Histonet]

I would second Terri's comments on the Sakura Glas coverslipper.  I do have to 
stress that yearly PM's are very important and that there is daily maintenance  
that needs to be done in order for the instrument to function properly, we also 
only use 24x50's.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: Terri Braud via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Friday, January 22, 2016 11:26 AM
To: 'histonet@lists.utsouthwestern.edu'
Subject: Re: [Histonet] best glass coverslipper

I've had experience with several glass coverslippers over the years.  While 
I've not tried the newest/latest/greatest of other brands, I've been running 
the Sakura Glas coverslipper for 8 years without a hitch.  It has been very 
dependable, with little downtime, and extremely easy maintenance.  We have 
averaged less than 1 service call per year since purchase, outside of the 
annual pm.  At the time we purchased it, it had the shortest set up and routine 
maintenance of the 4 big players that we tried.  It is definitely a more 
sensitive system than the tape. All glass systems are less forgiving of 
technical error than tape, but the results are fabulous.  We coverslip using 
only 24 X 50, so avoid the hassles of changing sizes.  
I hope this will help.  Sincerely, Terri

Terri L. Braud, HT(ASCP)
Anatomic Pathology Supervisor
Laboratory
Holy Redeemer Hospital
1648 Huntingdon Pike
Meadowbrook, PA 19046
ph: 215-938-3689
fax: 215-938-3874
   3. Best Glass Coverslipper? (Cooper, Brian)
Message: 3
Date: Thu, 21 Jan 2016 20:35:37 +
From: "Cooper, Brian"  Hey Histonet!

What's the best automatic glass coverslipper out there?  I only have experience 
with the Sakura tape slipper, which is AWESOME, but alas, we can't get one 
here.  Space is definitely an issue; would love to hear your experiences!

Thanks,

Brian D. Cooper, HT (ASCP)CM | Histology Supervisor Department of Pathology and 
Laboratory Medicine Children's Hospital Los Angeles
4650 Sunset Blvd MS#43- Los Angeles, CA 90027
Ph: 323.361.3357 Pager: 213-209-0184
bcoo...@chla.usc.edu



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[Histonet] NSH Awards and Scholarships

[Elizabeth Chlipala via Histonet]

Hello Histonetters

It's that time of the year again. Nominations for NSH Awards and Scholarships 
is open.

Histotechs do great work every day. What better way to honor those leaders in 
the field than by nominating them for an NSH award?  Check out all of the 
awards offered by NSH and nominate that 
deserving individual.

Liz
NSH Awards Chairperson




Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

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Re: [Histonet] mouse femur decal and paraffin embedding protocol

[Elizabeth Chlipala via Histonet]

James

We use 10% Formic Acid to decal mouse femur and tibia -  The rear legs are 
removed at the hip with a pair of rongeurs  the skin is removed to the ankle, 
we allow the knee joints to fix for 48 hours, they need to be in a normal 
degree of flexion, meaning you want them in a container that does  not bend the 
knee.  We trim off the muscle  and place in decal for overnight - process the 
next day on an hour processing cycle.  You don't need microwave for mouse knees 
they decal quickly.  Most of the work we do is for cartilage degeneration, 
rheumatoid or osteoarthritis, if you are looking at bone marrow I would modify 
slightly.  I can send a work instruction document that has more information in 
another e-mail if you are interested.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: Herrick, James L. (Jim), MSA via Histonet 
[mailto:histonet@lists.utsouthwestern.edu] 
Sent: Monday, January 11, 2016 10:40 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] mouse femur decal and paraffin embedding protocol

Dear Histonet Colleagues,

I hope the holiday season has been good to all.

We are interested in a routinely used protocol to decal, process and paraffin 
embed mouse femur and tibia. Some of our projects may require a gentle decal, 
whereas others may not be so delicate. Also, we do have access to a microwave 
for decalcifying, if recommended to be a better method. It would certainly save 
a lot of time.

It would be greatly appreciated if anyone would be willing to share a protocol 
for post-harvest sample storage (i.e. PBS, 70% ETOH, saline, etc.), 
decalcification, processing and embedding. Thank you in advance for your help!!

Jim Herrick, M.S.A.
Supervisor - Sr. Res.Tech.
Department of Orthopedics
Biomaterials and Histomorphometry Core Lab Biomaterials and Tissue Engineering 
Lab Office Phone: (507) 538-4300 Lab Phone: (507) 255-5946
Pager: 127 (13239)
Fax: (507) 266-9451
Email: herrick.ja...@mayo.edu
__
Mayo Clinic
200 First Street SW
Rochester, MN 55905
www.mayoclinic.org


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Re: [Histonet] IHC with H & E staining

[Elizabeth Chlipala via Histonet]

Marcus

You can stain with H after DAB if you think it would help your how you 
analyze your slides.  DAB with the addition of a special stain has been 
published and for us depending upon the project that we are working on we may 
choose to use a different counterstain after an IHC that has DAB as an 
chromogen.  For example we have worked with macrophage markers counterstained 
with Prussian blue, another example is brain IHC markers counterstained Cresyl 
ect Violet rather than hematoxylin.  DAB is extremely stable and therefore many 
special stains or different counterstains can be used.  

If you are running an image analysis algorithm most canned algorithms are set 
up to function with a hematoxylin counterstain - you would need evaluate if a 
different counterstain would decrease the accuracy of your algorithm but you 
may have the ability to design an algorithm with the counterstain of your 
choice.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 881-0763 cell
(303) 682-9060 fax
l...@premierlab.com

Ship to address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

From: Marcus Green via Histonet [histonet@lists.utsouthwestern.edu]
Sent: Wednesday, January 06, 2016 4:26 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] IHC with H & E staining

Dear Users,



Happy New year - I hope this finds everybody well!



I was asked a very simple question yesterday - why don't you do H 
counterstaining on DAB stained samples. The question came about as we're 
looking at CD31 staining for blood-vessels and some look ruptured (we're keen 
to see red blood cells).



Instead of doing a consecutive cuts, a CD31 stain (Haem counterstain) on one 
slide and H on another, the question was asked why not do the Eosin stain as 
part of the counterstain



I've never seen it done in the literature or in the clinic, and I've never 
asked why it's not done. Any assistance or advice would be greatly welcome - 
and my sincerest apologies if this is a very basic question?



Thanks in advance for your time,

kind regards,



Marcus,


Department of Oncology - University of Oxford,
Old Road Campus Research Building,
Roosevelt Drive,
Oxford,
OX3 7DQ


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Re: [Histonet] DAB IHC with H & E staining

[Elizabeth Chlipala via Histonet]

That's correct if you do not quench endogenous peroxidase activity completely 
you would be able to visualize the RBC's but maybe in instances like this it 
might be better to have the rbc's stain a different color than the DAB they 
would be easier to recognize, especially if they are specifically looking at 
neovascularization and subtle changes in the vasculature.  DAB can be great but 
it also can pose problems due to endogenous pigments that appear light brown or 
brown in color.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com<mailto:l...@premierlab.com>
www.premierlab.com<http://www.premierlab.com/>

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

From: David Wright [mailto:d...@uchicago.edu]
Sent: Wednesday, January 06, 2016 2:16 PM
To: histonet@lists.utsouthwestern.edu
Cc: Elizabeth Chlipala
Subject: RE: DAB IHC with H & E staining

Hi Histonet, Marcus and Liz

I agree with everything Liz says about precipitated DAB being wonderfully 
robust so that you can do a whole range of stains successfully after using it.

I am slightly puzzled that Marcus wants to reveal RBCs after a DAB reaction. 
Doesn't it just happen any way? Haem/hemoglobin provides a powerful 
non-specific peroxidase (the frothing you get with peroxide on dried blood) 
which will also precipitate DAB in the RBCs - I see it whenever I don't fully 
perfuse my brains. If you're not seeing this currently, and you are sure there 
are RBCs present, it might simply suffice to ease up on whatever procedure you 
use to "kill' endogenous peroxidases before your Ab incubations, so that the 
haem can go to work on the DAB.

Note that if you happen to use thick sections for floating immuno, you really 
don't want to use eosin. There is so much material that everything is red. 
(There is a similar 'too much' problem with using solochrome cyanine on thick 
sections for myelin...)

best to all - David
==
David A. Wright, Ph.D.
University of Chicago
Section of Neurosurgery, MC3026

ORIGINAL MESSAGES
--
Histonet Digest, Vol 146, Issue 4 Message: 7
Date: Wed, 6 Jan 2016 10:20:30 -0700
From: Elizabeth Chlipala <l...@premierlab.com<mailto:l...@premierlab.com>>
To: Marcus Green 
<marcus.gr...@oncology.ox.ac.uk<mailto:marcus.gr...@oncology.ox.ac.uk>>, 
"histonet@lists.utsouthwestern.edu<mailto:histonet@lists.utsouthwestern.edu>"
Subject: Re: [Histonet] IHC with H & E staining

Marcus

You can stain with H after DAB if you think it would help your how you 
analyze your slides.  DAB with the addition of a special stain has been 
published and for us depending upon the project that we are working on we may 
choose to use a different counterstain after an IHC that has DAB as an 
chromogen.  For example we have worked with macrophage markers counterstained 
with Prussian blue, another example is brain IHC markers counterstained Cresyl 
ect Violet rather than hematoxylin.  DAB is extremely stable and therefore many 
special stains or different counterstains can be used.

If you are running an image analysis algorithm most canned algorithms are set 
up to function with a hematoxylin counterstain - you would need evaluate if a 
different counterstain would decrease the accuracy of your algorithm but you 
may have the ability to design an algorithm with the counterstain of your 
choice.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
l...@premierlab.com<mailto:l...@premierlab.com>

From: Marcus Green via Histonet [histonet@lists.utsouthwestern.edu]
Sent: Wednesday, January 06, 2016 4:26 AM
To: histonet@lists.utsouthwestern.edu<mailto:histonet@lists.utsouthwestern.edu>
Subject: [Histonet] IHC with H & E staining

Dear Users,

Happy New year - I hope this finds everybody well!

I was asked a very simple question yesterday - why don't you do H 
counterstaining on DAB stained samples. The question came about as we're 
looking at CD31 staining for blood-vessels and some look ruptured (we're keen 
to see red blood cells).

Instead of doing a consecutive cuts, a CD31 stain (Haem counterstain) on one 
slide and H on another, the question was asked why not do the Eosin stain as 
part of the counterstain

I've never seen it done in the literature or in the clinic, and I've never 
asked why it's not done. Any assistance or advice would be greatly welcome - 
and my sincerest apologies if this is a very basic question?

Thanks in advance for your time,

kind regards,
Marcus,
Department of Oncology - University of Oxford,
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Re: [Histonet] VIP Model E150/E300

[Elizabeth Chlipala via Histonet]

I have an operator/user manual that I can pdf for you but I do not have the 
service manual.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: Adesupo, Adesuyi (Banjo) via Histonet 
[mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, January 05, 2016 5:28 AM
To: 'histonet@lists.utsouthwestern.edu'
Subject: [Histonet] VIP Model E150/E300



Hi



Does anyone has a service manual for the VIP Model E150/E300 and would like 
to share with me?  I will greatly appreciate it.




   Best regards,

   Banjo Adesuyi, BMLS, HT (ASCP) HTL, QIHC, QLS
   Histology Supervisor
   Norman Regional Health System,
   Norman, OK 73071.
  Tel: 405- 307- 1145
  abades...@nrh-ok.com

==
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Re: [Histonet] Mouse Brain Processing Help

[Elizabeth Chlipala via Histonet]

Laura

They may not be processed enough.  We process brain samples for both mouse and 
rat 2 mm thick sections (cut on a brain matrix) on a 45 minute cycle.  If they 
are processing half of the mouse brain (these samples can be 4 -5 microns 
thick).  I don't think the cycle is long enough, that is why the blocks are 
swelling up when they are trimmed and placed on ice.  A well processed brain 
block should not swell up when it is placed on ice.  The separation of the 
brain tissue from the paraffin on the water bath is also a sign that the 
samples are under processed.

Here is the cycle we use for brain samples - you may need to adjust this a bit 
for your samples but I think if you process a bit longer they might have better 
success with sectioning.  Brain can be tricky.  We have the ability to add an 
extra 100%, xylene, and paraffin step to this cycle if necessary.  For mouse 
tissue (unless its skin, bone and brain) our cycle is normally 20 minutes per 
station,  but we have found that brain does need to be processed a bit longer.  

50% alcohol - 45 minutes
70% alcohol - 45 minutes
80% alcohol - 45 minutes
95% alcohol - 45 minutes
100% alcohol - 45 minutes
100% alcohol - 45 minutes
Xylene - 45 minutes
Xylene - 45 minutes
Paraffin - 45 minutes
Paraffin - 45 minutes
Paraffin - 45 minutes

Good Luck

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

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Longmont, CO 80504

-Original Message-
From: Laura Tarwater via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, December 02, 2015 10:40 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Mouse Brain Processing Help

I need some advice on processing mouse brain.  We have an outside lab using our 
automatic processor and embedding equipment and has been having issues section 
their mouse brain.  I was informed that the sections are extremely brittle and 
rolling but mostly just the paraffin and not the tissue itself.  They are 
seeing the brain tissue separate from the paraffin once it is the water bath.

Had better results when placing a soaked gauze on the surface of the block 
instead of chilling or soaking block to prevent swelling of tissue.

Here is the protocol they are using for harvesting and processing:
1.  Tissue perfused to 4% PFA at time of sacrifice
 Brain is cut in half at this point and separated 2.  Tissue placed in 
cassette in 10% NBF for 24hrs 3.  Then placed in 70% ETOH until able to process 
(usually the next day) 4.  Processing Schedule (all done with vacuum)
   30min 70%  ETOH
   30min 80%  ETOH
   30min 90%  ETOH
   30min 100% ETOH
   30min 100% ETOH
   30min 100% ETOH
   30min xylene
   30min xylen
   5min empty tank to remove excess xylene
   30min paraffin
   30min paraffin

I am not a histotech so any advice you can give me would be great.

Thanks,


*Laura Tarwater*

*Tissue Bank Technician*

*Harper Cancer Research Institute*


*University of Notre Dame574-631-2562*
*tarwate...@nd.edu *
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Re: [Histonet] Recommendations of places to purchase two stains and question on protocol

[Elizabeth Chlipala via Histonet]

Jorge

I can comment on the cresyl etch violet - we actually purchased  cresyl violet 
acetate from MP Biomedicals.  This particular chemical has not been certified 
by the biological stain commission, if you are looking for a dye that has been 
certified then I would go to Sigma-Aldrich - they also supply this chemical.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Jorge A. Santiago-Blay via Histonet 
[mailto:histonet@lists.utsouthwestern.edu] 
Sent: Friday, November 27, 2015 9:58 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Recommendations of places to purchase two stains and 
question on protocol

Dear Histonetters:


I am trying to purchase two stains:

1. Cresil etch violet (for nervous tissue) - 
https://en.wikipedia.org/wiki/Cresyl_violet




2. Mallory azan (for encocrine tissue)  - 
https://www.emsdiasum.com/microscopy/technical/datasheet/26450.aspx

 ,

 https://en.wikipedia.org/wiki/Heidenhain%27s_AZAN_trichrome_stain


Have searched the web and rather than gamble, wish to know if I could receive 
recommendations on good places to purchase this type of reagent.
Good customer service and affordable prices a must.

In both cases, do you know if these stains work on tissues preserved in ethanol 
(70%), some of the tissues could have been preserved for years.

If you have constructive feedback, please feel free to email me at

blayjo...@gmail.com

Gratefully,

Jorge


Jorge A. Santiago-Blay, PhD
blaypublishers.com

1. Positive experiences for authors of papers published in *LEB* 
http://blaypublishers.com/testimonials/

2. Free examples of papers published in *LEB*:
http://blaypublishers.com/category/previous-issues/.

3. *Guidelines for Authors* and page charges of *LEB*:
http://blaypublishers.com/archives/ *.*

4. Want to subscribe to *LEB*? http://blaypublishers.com/subscriptions/


http://blayjorge.wordpress.com/
http://paleobiology.si.edu/staff/individuals/santiagoblay.cfm
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Re: [Histonet] DIF on paraffin embedded tissue

[Elizabeth Chlipala via Histonet]

I'm not sure that is the case in the grand scheme of things, it will depend 
upon the target that you need to stain via immunofluorescence.  Technically we 
perform IF staining on frozen sections primarily because the antigen does not 
survive formalin fixation.  Many people utilize IF techniques on FFPE tissue 
with good success, we have done it here.  There are things you will need to 
consider and what I suggest below may not work at all.  It's up to you if you 
want to try it or not and you may feel it is not worth the time and energy 
required to see if it may work on FFPE samples, it could be a lot of work and 
it may not be successful.   

1.  You will likely not be able to use your current protocol
2.  You will likely need to add an antigen retrieval step
3.  You may need to look for a different antibody source (one that survives 
formalin fixation)
4.  The signal needs to be good since formalin fixation will cause 
autofluorescence 

Unless I am completely missing something here since two individuals have stated 
that the sample is useless, maybe there is a better explanation as to why the 
sample is completely useless.  Here is where I am coming from - technically you 
can perform IF staining on FFPE tissue samples, people do it all of the time, 
we have done it here, it's a common technique for dual labeling of samples when 
co-localization is an expected result.

Good Luck

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Simmons, Christopher via Histonet 
[mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, November 25, 2015 9:10 AM
To: Rene J Buesa
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] DIF on paraffin embedded tissue

Useless sample
Sorry

Sent from my iPhone

> On Nov 25, 2015, at 11:02 AM, Rene J Buesa via Histonet 
>  wrote:
> 
> No matter WHO to tell you to do WHAT, for IF purposes, that FFPE 
> tissue is USELESS.René
> 
> 
>On Wednesday, November 25, 2015 10:55 AM, Maryann Deathridge via Histonet 
>  wrote:
> 
> 
> We have a tissue sample that was processed and paraffin embedded.  We 
> URGENTLY need to recover the tissue and perform Immunofluorescence on 
> the sample.
> Does anyone have a procedure.  HELP
>   
> madeathri...@pastnashville.com
> 
>   
> 
> 
> 
> 
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Re: [Histonet] DIF on paraffin embedded tissue

[Elizabeth Chlipala via Histonet]

Teri

Excellent point and to add to that most IF techniques that are employed on FFPE 
are not direct they are indirect and consist of an unlabeled primary and then a 
secondary that is conjugated with an alexa fluor or something similar at least 
that is how we approach IF on FFPE tissues here.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: Teri Johnson via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, November 25, 2015 11:30 AM
To: 'histonet@lists.utsouthwestern.edu'
Subject: Re: [Histonet] DIF on paraffin embedded tissue

Maryann,

Whoa, bad day. I'm sorry for you and for the patient.

As for whether the testing usually performed in frozen section by Direct 
ImmunoFluorescence can be successfully done on FFPE, Liz Chlipala is probably 
the closest to answering this. This was likely a kidney or skin biopsy. The 
panel is likely something like IgG, IgA, IgM, C3, Fibrinogen and maybe another 
marker or so.

Can it be done? I would say not with any degree of confidence unless this was 
extensively tested side-by-side with frozen vs paraffin embedded tissues. The 
IHC stain techniques would be radically different. This would take a lot of 
time.

Now, if you were successful in providing these assays that you have validated 
in your laboratory (I shudder to think what that would entail), would the 
pathologist be comfortable signing out a case such as this? Would the clinician 
be comfortable treating the patient based on these results?

Teri Johnson
Manager, Clinical Trial Testing
Genoptix, Inc., a Novartis company
BioPharma
1811 Aston Avenue
Carlsbad, CA  92008
USA

Phone +1 760 516 5954
tejohn...@genoptix.com
www.genoptix.com




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Re: [Histonet] Do you check humidity and room temp for your daily QC in the lab?

[Elizabeth Chlipala via Histonet]

We do both, we only have criteria for the temp but we do record both in all 
rooms in the laboratory.  We use traceable wall thermometers/humidity units and 
replace upon expiration.   We have a few SOP's that govern this, we are a GLP 
compliant lab.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: Histology Technician via Histonet 
[mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, November 12, 2015 10:17 AM
To: Histonet List
Subject: [Histonet] Do you check humidity and room temp for your daily QC in 
the lab?

 Does anyone check humidity and room temp as your QC?  If so, can you share you 
procedure and QC form?  Do you use a company or do you monitor it yourself?
Thanks!
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Re: [Histonet] Temperature logging

[Elizabeth Chlipala via Histonet]

Karen

There was a similar question on temp charts the other day on the histonet.  We 
record our temps daily on a form.  Our temperature range is determined by the 
equipment in the room, all operating manuals have ranges listed for temp, we 
list all major equipment in a room and what the recommended temperature range 
is listed in the operating manual and make a determination of a range based 
upon how critical the equipment is, etc.  We have an SOP that governs this and 
how we manage the thermometers also.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Heckford, Karen - SMMC-SF via Histonet 
[mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, October 01, 2015 11:43 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Temperature logging

Good Morning,
I have been asked to monitor the room temperatures in the Histology lab, and 
anywhere we store paraffin blocks, slides and reagents.  Some of you I know 
already do this.

1st Question:   What is your temperature range?
2nd Question:  Are you physically monitoring it daily and writing it down on a 
temperature chart?

The reason why Question 2 was I purchased these new thermometers that I can put 
in the Hi and Lo.  It also has where I can download it on to a scan disk so I 
can down load and put on a flash drive or store in the computer.  I am not sure 
why these will not work but I am getting grief from my Lab Director that is a 
Med Tech.  I figure if it alarms on me I can down load it and figure out the 
time of the out of range since I am not here on the weekend.  Cannot have 
anyone else do it because I am the only Histology person in this hospital.  The 
lab people do not give much support when a alarm is going off they ignore it.


Karen Heckford HT ASCP CE
Lead Histology Technician
St. Mary's Medical Center
450 Stanyan St.
San Francisco, Ca. 94117
415-668-1000 ext. 6167
karen.heckf...@dignityhealth.org

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Re: [Histonet] Hematoxylin Precipitate

[Elizabeth Chlipala via Histonet]

I think you may a have a serious problem if "so much tissue washes off"  if 
that is happening  you have problems with tissue adherence.  A properly 
processed and cut section should not wash off the slide, or even a portion of 
it should not wash off.  That would mean that you did not provide to the 
pathologist what is represented in the block.  If that is the case then you 
would need to filter all solutions on a stainer daily not just the hematoxylin. 
 

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: Tim H via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, September 22, 2015 11:25 AM
To: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Hematoxylin Precipitate

You should be filtering your Hematoxylin on a daily basis regardless of what 
the manufactures says.  We use to filter twice a day since we did a traditional 
overnight run and then again in the afternoon for specimens that had been 
microwave processed.  So much tissue washes off in the solutions they should be 
changed or filtered fairly regularly to try and prevent cross contamination on 
the slides.
 
You can also try increasing your rinse times and see if that doesn't help as 
well.  
 
Thanks,
 
Tim
> 
> Message: 1
> Date: Mon, 21 Sep 2015 15:14:39 -0500
> From: "Sandra Cheasty" 
> To: "Histonet (histonet@lists.utsouthwestern.edu)"
>   
> Subject: [Histonet] Hematoxylin Precipitate
> Message-ID: <4cda87133587e64c965ce6c356d18...@svm.vetmed.wisc.edu>
> Content-Type: text/plain; charset="us-ascii"
> 
> Hello all,
> 
> Has anyone using Richard Allen Hematoxylin-2 noticed an odd 
> artifact on the slides after using the Hematoxylin for more than a few days 
> on their stainer? We are seeing small spore or pollen-like blue dots here and 
> there on the slides. It is not coming from the water bath or our water supply 
> on the stainer. I used sterile gloves, opened a new case of slides, dipped 
> them in DI water, then in the RA Hematoxylin 2 on the stainer, then in DI 
> again, air-dried and coverslipped them, and the blue dots were there. The 
> only way we got rid of the blue artifact was to use new RA Hematoxylin-2 
> every 2-3 days, which is a bit expensive.
> 
> Thanks for your input, and if you can recommend a different, 
> reasonably priced hematoxylin, that would be awesome.
> 
> Cheers,
> 
> Sandy
> 
>  
> 
> Sandra J. Cheasty, HT (ASCP)
> 
> Histology & Necropsy Supervisor
> 
> UW-Madison, School of Veterinary Medicine

  
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Re: [Histonet] Hematoxylin Precipitate

[Elizabeth Chlipala via Histonet]

Tim

First of all my comment was not meant to criticize your post and even if you 
may not think so I was trying to help.  I stand by what I said, my comment was 
to address what I thought was the amount of tissue loss your lab experiences 
and if I took your comment too literally I apologize but I firmly believe that 
properly processed and sectioned tissue samples should remain on the slide.  I 
do understand that we will on occasion have loss of tissue from the slides that 
we cut and stain.   We will see a lens floating in one of the alcohols when we 
stain mouse eyes or a portion of a dermal construct will come off the slides if 
we have not dried it properly.  Most of the tissue loss we experience is due to 
improperly processed and sectioned samples.   

We are a small lab and we are GLP compliant.  We do not change our H staining 
reagents daily our volume varies depending upon the projects we are working on, 
but I can tell you that we have looked at H staining over time and we have 
made sure that our reagents are changed appropriately.  We do not filter our 
hematoxylin daily and have not experienced many floaters or carry over or other 
things on our slides.  

Liz from wacky tabacky country

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: Tim H via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, September 22, 2015 3:04 PM
To: gayle.cal...@bresnan.net; Histonet
Subject: Re: [Histonet] Hematoxylin Precipitate

Liz, Phillip and all that are
interested,

 

I take it you have guys never looked at or had someone else examine what is at 
the bottom of the Hematoxylin filter after you put through a day's work.  There 
will be tissue particles of tissue along with other contaminates, I am not 
saying you are going to see a complete LEEP sitting at the bottom but you will 
have contamination.  

 

Liz, maybe the tissue super adheres up there in wacky tabacky country and 
Phillip sitting in a research facility (are you even involved with processing 
and staining of slides or just publishing
articles?) but in the rest of the United States, I can guarantee you there will 
be some tissue in the Hematoxylin if you use a traditional dip and dunk system. 
 

Liz, you do bring up a good point
about having tissue in every container. To a degree you will have tissue in 
every reagent container. I was assuming and maybe unjustly that most labs are 
using Good Laboratory Practice and discard their reagents after they have used 
them for a staining session.  This topic was about Hematoxylin Precipitate and 
"small spore or pollen-like blue dots" which I did not say was tissue, I was 
passing along some of my 23 years of knowledge.

Listen; don't take my word for
it.  You can have Ventana come out to your facility and filter all your 
reagents and stains and have them tell you what is in your solutions.
Next time just pass along good
information and criticize those trying to help!  To many people like to make a 
negative on this site of people trying to truly help.  This is why I rarely 
post on Histonet but instead directly email the person.

By no means am I promoting
Ventana/Roche products.  I am just passing along information that might be 
helpful.  

 Tim Disclaimer: This information is by no means is meant for any weak stomach 
individuals or those preparing for the  zombie apocalypse.   
 
> From: gayle.cal...@bresnan.net
> To: thiggin...@msn.com; histonet@lists.utsouthwestern.edu
> Subject: RE: [Histonet] Hematoxylin Precipitate
> Date: Tue, 22 Sep 2015 12:27:21 -0600
> 
> Sandy,
> 
> After years of using Richard Allan's hematoxylin 2 with great success,   if
> we didn't filter daily before use, we had stain precipitate on sections.
> Some of this comes from the hematoxylin continuing to oxidize in open air,
> bacteria and other "crud".   Tim is absolutely correct ignoring
> manufacturers no filtering instructions.   Being old school, we were taught
> to faithfully filter any hematoxylin, regardless of progressive or
> regressive types.If we topped off hematoxylin 2 or used new stock,  the
> stain was filtered into a CLEAN staining container/dish.  Keep an extra
> container around if possible.   We used a medium fast filter paper, Whatman
> 54.   I realize this takes time but junk on a slide is NOT good thing,
> especially after IHC staining and have a photo to show this - the result of
> being lazy and not filtering the hematoxylin on that particular day.   
> 
> We used a distilled water rinse before hematoxylin2, but DI H2O will 
> be contaminated with cellular debris and last hydrating alcohol carryover.
> Change DI water frequently if you have many runs in a day.   We used 1
> minute running tap water 

Re: [Histonet] DAKO LINK PTs

[Elizabeth Chlipala via Histonet]

Tiana

We have never experienced any issue like that here we will use the HIER 
solutions in the PT links for up to three times within a period of 2 weeks.   
We have had our PT link units validated and they are calibrated yearly.  Do you 
review the Target Retrieval Run Reports, we print and keep all of ours.  Our 
units are primarily programed to heat to 80C we put the slides in, warm up to 
95C retrieve for 20 minutes cool down to 80C remove slides right after that.  I 
would think if you left slides in the retrieval solution for different times 
after they are completed you might see some changes in staining intensity, that 
why we try to be consistent and remove the slides as soon as they are 
completed.  We do use other retrieval times and temps on some occasions but 
what I stated is our standard protocol.   I hope this helps.  

There is one other thing, it think it is extremely important to clean the 
instrument as it is required, we have in our SOP's after 200 slides I don't 
know what Dako recommends but we actually clean more often typically after 
around 80 to 120 slides or so.  That will keep your probe nice and clean and 
decrease your variability, if you don't clean you can get build up in the probe 
and that is just going to cause inconsistent staining, its easy to set up a 
cleaning cycle at the end of the day.

There are also a lot of other factors that can affect staining consistency - 
tissue placement on the slide in one thing, we place our tissue in the same 
area on the slide, same number of drop zones, appropriate amount of reagents, 
cutting corners on amount of reagents will not lead to consistent high quality 
staining.Section thickness can also lead to variation in staining 
intensity.  

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: Tiana Baskin via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, July 14, 2015 8:42 AM
To: 'histonet@lists.utsouthwestern.edu'
Subject: [Histonet] DAKO LINK PTs

Hi Histonetters,

I have encountered a problem with some of my staining and I am struggling to 
pinpoint the root cause. I was wondering if any other DAKO Autostainer Link/PT 
users are experiencing the same oddities.

It seems like the first runs with fresh HIER PT solution is very typical of 
what we have optimized and the second batch of slides has more background and 
in some cases nuclear staining (especially in Actin (1A4) and pan CK (AE1/AE3)) 
when the target is cytoplasmic. We do not use the PT solution more than twice. 
What do others do? Have you seen similar things?

Tiana


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Re: [Histonet] Trichrome troubleshooting

[Elizabeth Chlipala]

Suzanne

How many times have you used the kit and reagents, I did look up how the kit 
works but the trichrome stain can be tricky.  First of all you need to make 
sure that the mordant (bouins solution) is at 60C prior to placing your slides 
in them.  We normally heat up our bouins for at least an hour prior to placing 
the slides in the solution.  We leave in bouins for an hour and a half rather 
than an hour.   I see that this is a microwave protocol I cannot comment on 
that but I don't think that the hematoxylin is the issue, if you leave longer 
in 1% acetic acid that may pull some of the blue stain out or I would try 
dehydrating with lower alcohol percentage that can pull some of the blue stain 
out.   I would also try leaving it a bit longer in the bouins after you 
microwave it - that might help.

Trichrome staining works best with fresh reagents so if you have used these 
reagents too much that could cause problems.  I'm also not a big fan of the one 
step trichromes, they are quicker but sometimes not as good as the two steps, 
just my opinion.

FYI - to evaluate your staining look for a smaller vessel, the smooth muscle 
should be nice a red, if its greyish or blue you have not done the stain 
properly.  Good Luck

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: Suzanne Martin [mailto:smar...@lcpath.com] 
Sent: Tuesday, June 30, 2015 12:37 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Trichrome troubleshooting


Hi all,

We are having trouble troubleshooting our trichrome. It is too blue. We are 
using Leica's kit with the Weigerts iron with Gills. Most of the small bowel 
controls have seen improvement but patient tissue is not... strange. 

We have tried lessening the time in Gills, adding time for the last acid step, 
even lessening time and adding time in the Weigerts. 


Thoughts?

Thank you.

Suzanne HT




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Re: [Histonet] OJT Histotechs/Training

[Elizabeth Chlipala]

Experience may not be the issue she needs to have an associate's degree with 
the appropriate credit hours to sit for the HT.  It does not matter where you 
get your experience it does not need to be in a clinical lab.  I have had 
several techs sit for the HTL registry and IHC qualification with research 
experience and I myself signed for their experience.  I also needed to write a 
supplemental letter to the BOR explaining my position and that as a contract 
histology based CRO we did not have a medical director, etc.

Here is essentially what I wrote to the BOR this was back in 2004, this was for 
the IHC qualification but I had written a similar letter to the BOR for her HTL 
certification, I also wrote one for myself for the IHC qualification when I 
took that back in 2004 also.

I'm writing this letter to request a special consideration for the 
Immunohistochemistry Qualification for my employee Amy XX.  She has 
registered to take the Immunohistochemistry Qualification this spring.  She has 
already received the information packet.  I own a small contract histology 
laboratory called Premier Laboratory, LLC.  I have attached copies of our 
Articles of Incorporation, Colorado Division of Workers' Compensation, IRS 
Employer Identification Number, Colorado Business Registration and Application 
for Employer Identification Number.  We are a small lab, consisting of myself 
and Amy.  We do not have a PhD, MD, or DVM on staff to vouch for myself or 
Amy's level of experience performing immunohistochemical stains.  

Amy has been employed by Premier Laboratory since Feb 1, 2003.  Amy also worked 
for me at BolderPATH (another business that I was a part owner) from June 1, 
2002 to Jan 31, 2003.  During her employment at BolderPATH and Premier 
Laboratory  the majority of her time was spent developing immunohistochemical 
protocols and performing various immunohistochemical applications. Some of the 
antibodies that Amy has created protocols for and performed for clients at 
Premier Laboratory include:

BrdU, Ki-67, PCNA, cleaved caspase-3, GFAP, CD31, F4/80 (clone BM8), F4/80 
(clone A3-1), complement C3, Crry/p65, GAP-43, Luciferase, NOS2, PSA, 
synaptophysin, smooth muscle actin, S-100, LCA, CD20, UchL-1, CD11b, CD14, 
CD13, CD105, MMP-1, MMP-7, FLK-1,  plus several in-house antibodies for various 
applications.  I have performed these antibodies utilizing a variety of 
fixatives, processing methods and detection systems utilizing both frozen and 
paraffin embedded material.

I have attached to this letter the Immunohistochemistry (IHC) Qualification 
Reference, which I have filled out for Amy.

If you have any questions or require any additional information please give 
myself or Amy a call at 

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: Coffey, Anna (NIH/NCI) [C] [mailto:anna.cof...@nih.gov] 
Sent: Tuesday, May 19, 2015 12:01 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] OJT Histotechs/Training

Hi Loralei,



Maybe I'm just misunderstanding, but I'm unclear why you would be ineligible to 
sit for the exam with your experience. I've always worked in a research lab on 
animal tissues and used that experience to qualify for the exam. There is no 
longer a practical portion of the exam (the entire thing is a multiple choice 
electronic exam) and none of the paperwork requires a pathologist to sign off 
as your supervisor (this is the form to verify experience: 
http://www.ascp.org/pdf/BOC-PDFs/Documentation-forms/DocumentationFormHTLRoute2.aspx).
 According to the ASCP site, experience counts as one year full time 
acceptable experience in a histopathology (clinical, veterinary, industry or 
research) laboratory in the U.S., Canada or an accredited laboratory* within 
the last ten years.



It's my understanding that the exam requirements have changed over the last few 
years (before I got my certification) and maybe you now would be eligible for 
the certification?

Anna Coffey, MS, HTL(ASCP)CM
Histotechnologist
Center for Advanced Preclinical Research Frederick National Laboratory for 
Cancer Research Leidos Biomedical Research, Inc.
Bld 539, 224
Frederick, Maryland 21702
anna.cof...@nih.gov
301-846-1730





Message: 3

Date: Mon, 18 May 2015 11:59:59 -0700

From: Loralei Dewe lld...@gmail.commailto:lld...@gmail.com

To: Joelle Weaver joellewea...@hotmail.commailto:joellewea...@hotmail.com

Cc: Jennifer MacDonald jmacdon...@mtsac.edumailto:jmacdon...@mtsac.edu, 
Mayer,  Toysha N

tnma...@mdanderson.orgmailto:tnma...@mdanderson.org, 
histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu

[Histonet] NSH - 2015 Awards Banquet

[Elizabeth Chlipala]

Hello Everyone

It's that time of the year again.  As you know this year's NSH Convention is 
being held in Washington DC.  The Stars  Stripes NSH Awards Ceremony  
Celebration will be on Saturday August 28th.

The NSH offers many awards, too numerous to list here, so to see the list of 
awards available head to the NSH website http://nsh.org/scholarships-awards. 
There are three categories for awards; leadership, education and advocacy.   
These awards offer financial incentives for continuing education along with 
recognizing the individuals within our profession who have demonstrated both 
dedication and excellence in the field of Histotechnology.  I can't stress to 
you enough how great it is that the NSH can provide this much support to their 
members for continuing education opportunities. These opportunities are 
provided through the generosity of the sponsors, if it were not for them we 
would not have these awards.

It's also so important that we take time out of our busy days to nominate 
individuals in our field that are raising the bar and setting a higher 
standard, these individuals need and should be recognized and what a better way 
to do that then to nominate them for an award.  I challenge all of you to take 
the time to nominate one individual you feel is deserving of one of the many 
awards the NSH has to offer.

If you have questions or need help in nominating a colleague or yourself please 
feel free to contact me at l...@premierlab.commailto:l...@premierlab.com.

Thanks, have a great day and nominate.
Liz
NSH Awards Chairperson

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.commailto:l...@premierlab.com
www.premierlab.comhttp://www.premierlab.com/

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

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RE: [Histonet] prevent wrinkles when cutting

[Elizabeth Chlipala]

Rachel

We also run multiple antibodies under multiple parameters and there is no need 
or reason to cut in one ribbon that many sections.  You will still be able 
collect somewhat serial or the sections that you need with multiple ribbons.  
You are only able to fit on the waterbath in one ribbon (depending upon the 
size base mold that you used ) anywhere from 6 to 15 sections.  You can place 
ribbons side by side on the waterbath but for me personally I find it’s a bit 
more difficult to pick the sections up once you have placed multiple long 
ribbons on the waterbath.  I prefer working with one ribbon at a time, two tops 
when collecting multiple sections, but that’s just me.   We work with animal 
tissue and tissue that may not have been processed in our lab so we do soak all 
of the blocks.

The other thing you need to consider is that when we section a paraffin block 
the section thickness is not exactly consistent, we are essentially cutting a 
wedge as the block warms the sections becomes thicker, so if we can control the 
number of sections we collect in one ribbon, place the block back on ice before 
we collect the next ribbon, we can then maintain section thickness better.   I 
would think that as you section 30 +sections in one ribbon the block is warming 
up and the sections are becoming thicker.  Section thickness is extremely 
important when comparing IHC staining results, staining intensity will change 
in a 1 micron difference in section thickness.

Just my two cents - and I do have images and staining intensity calculations 
with respects to section thickness.  This is something that we have looked at 
recently.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Rachel M 
Gonzalez
Sent: Monday, April 20, 2015 3:05 PM
To: Joelle Weaver
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] prevent wrinkles when cutting

Hi

The long ribbons are for RD and QA purposes. Often we test under multiple 
conditions 1-4 with multiple antibodies to the same tissue or multiple tissue 
to optimize reagents.  It quickly adds up.

Rachel


On Mon, Apr 20, 2015 at 3:28 PM, Joelle Weaver joellewea...@hotmail.com
wrote:

 Why are you cutting such a long ribbon? You usually only a need a 
 series of 3-4 sections even for ribbon cutting. Might be easier to 
 control if you don't try to move such a long ribbon to the waterbath. 
 Drag the shorter ribbon towards you on the waterbath. Make sure the water is 
 not too cool.
 Face the block to full face but superficial, chill on ice for some 
 moisture, take sections while the block is still very cold. Use a 
 slow, steady, smooth stroke if doing manual cutting. Make sure your 
 embedding works well for the way you orient the block in the holder. 
 Angles work well for many tissues that are prone to wrinkling. Its 
 mostly just practice though. The more you cut, the easier it becomes 
 and usually the better you get at it.


 Joelle Weaver MAOM, HTL (ASCP) QIHC





  Date: Mon, 20 Apr 2015 19:06:17 +0200
  From: j.benavi...@eae.csic.es
  To: histonet@lists.utsouthwestern.edu
  Subject: Re: FW: [Histonet] prevent wrinkles when cutting
 
  Hi there,
 
  I´m curious about the soaking thing. We have never done it in our lab.
  Which is the purpose to do it?
 
  Than, after facing the blocks, we chill them in a cold plate so, if 
  wanting to do the soaking , when should we? I guess before placing 
  them on the cold plate, but that may cause a bit of ice formation?
 
  Thanks a lot for your help
 
  Julio
 
 
  On 20/04/2015 18:40, Grantham, Andrea L - (algranth) wrote:
   Rachel,
   First off, are you chilling and soaking the blocks after you face them?
   Do that and see if there is a difference.
   Don't try to get many sections to your ribbons. Shoot for a 
   smaller
 ribbon (5-6) sections that are good. Cut slowly but consistently.
   What microtome are you using? Are you using disposable blades and 
   are
 they sharp? Don't expect them to cut well if you use the same blade to 
 face the blocks. If you aren't using disposables, get some! They will 
 make your life easier.
   You might try to find a histotech at a local hospital lab who 
   might be
 able to give you a hands-on lesson.
   Don't despair! We all sat down at our microtomes those first times 
   and
 suffered trying to get perfect sections. It takes practice. You might 
 make some blank blocks or blocks with tissue you can spare to practice 
 your cutting techniques. I used to do this with my students and it 
 really helped them.
   Good luck!
  
   Andi G.
   

RE: [Histonet] Maximum size of tissue for paraffin processing

[Elizabeth Chlipala]

We processed tissues as large as elk and lion vocal cords - 2 cm x 5 cm.  You 
just need to adjust the time accordingly.  Very large samples we would process 
manually through graded alcohols and then the last absolute, xylene and 
paraffin would be run on the tissue processor, we have programs that range from 
6 to 24 hours a station depending upon tissue size.  You can run the entire 
process cycle on a tissue processor its just that we did not want to hang up 
one of the processors for a week on one study.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Caroline Miller
Sent: Thursday, April 09, 2015 12:26 PM
To: Histonet@Lists. Edu
Subject: [Histonet] Maximum size of tissue for paraffin processing

Hi there wonderful Histonet people,

Has anyone seen any studies on the maximum size of tissue that can be paraffin 
(or resin) processed. I am not talking about the size that can fit in a tissue 
cassette, but, for example, entire pig bladder processing.

I realize it would include extended processing time, temp, vacuum, agitation so 
imagine that none of these factors were limiting as I shall be using an 
automated tissue processor.

I also realize that this is going to be tissue dependent. I was hoping there 
were already some studies in this area that I am not finding in both google and 
pubmed searches.

thanks, in advance, for any links you can point me to,

yours,
Caroline

--
Caroline Miller
Director of Histology
3Scan.com
415 2187297
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RE: [Histonet] type IV collagen antibody

[Elizabeth Chlipala]

Amy



It’s been some time but we have had success in the past with the following 
antibodies – the SouthernBio and Abcam antibodies listed below


Antibody

Vendor

Catalog Number

Clone

Host

Hu

Rat

Rab

Ms

Pm

Sh

Gt

Pig

GP

Dog

Comments

Collagen IV

SouternBio

0340-01

UNLB

Goat

X





X











X



Collagen IV

abcam

ab6586

Poly

Rabbit

X





X


















Good Luck



Liz



Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC

Premier Laboratory, LLC

PO Box 18592

Boulder, CO 80308

(303) 682-3949 office

(303) 682-9060 fax

(303) 881-0763 cell

l...@premierlab.com

www.premierlab.com



March 10, 2014 is Histotechnology Professionals Day



Ship to Address:



Premier Laboratory, LLC

1567 Skyway Drive, Unit E

Longmont, CO 80504





-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Amy Lee
Sent: Tuesday, April 07, 2015 3:33 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] type IV collagen antibody



Hello histonet,



Could anyone recommend a  type IV collagen antibody work on FFPE mouse tissue?



Thanks in advance,



Amy

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[Histonet] RE: Question about Formic acid decal and TRAP stain

[Elizabeth Chlipala]

Debbie

In our hands it has not worked on formic acid decaled samples, EDTA decal only.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Debra Siena
Sent: Thursday, April 02, 2015 10:00 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Question about Formic acid decal and TRAP stain

Hi Histonetters,

I have a question to ask if you don't mind.  Can TRAP Histochemical staining be 
performed after decalcifying with formic acid? Any tricks of the trade, etc?  
If anyone has any experience or references that they could point me to, I would 
greatly appreciate it.  Thanks in advance for your help.




Debbie Siena
dsi...@statlab.com mailto:bbro...@statlab.com%7C | 
www.statlab.comhttp://www.statlab.com/

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RE: [Histonet] Tol Blue

[Elizabeth Chlipala]

On occasion you might lose the metachromatic staining with T. Blue if you if 
you dehydrate in alcohol.  We normally air dry, place in xylene and coverslip.  
You will never run into a problem with the staining if you do that.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jennifer 
MacDonald
Sent: Tuesday, March 24, 2015 3:55 PM
To: Kimberly Marshall
Cc: histonet@lists.utsouthwestern.edu; histonet-boun...@lists.utsouthwestern.edu
Subject: Re: [Histonet] Tol Blue

Mast cells granules are metachromatic.  What you see is the expected staining 
reaction.



From:   Kimberly Marshall kimbe...@animalreferencepathology.com
To: histonet@lists.utsouthwestern.edu 
histonet@lists.utsouthwestern.edu
Date:   03/24/2015 02:20 PM
Subject:[Histonet] Tol Blue
Sent by:histonet-boun...@lists.utsouthwestern.edu



?Hello my fellow Histo Techs.  Have a question I just know someone out 
there can answer for me.

In canine tissue, we are having problems with the Tol Blue for mast cell. 
Am experiencing metachromasia, or the mast cells turning purple not blue. 
I have attempted to decrease time, or add time, but its not helping.  My 
pathologist says he has had this issue before.  So question is.  Could it 
be the mast cell in a dog does not stain the same? Is there another stain 
that may work?  Any help will be much appreciated.


Thanks in Advance.

Kimberly Marshall H.T.(ASCP)






Kimberly Marshall H.T.(ASCP)

Histology/Lab Supervisor

Toll Free 1-800-426-2099

Fax 801-584-5104

PO Box 17580

Salt Lake City, Utah 84107

www.animalreferencepathology.comhttp://www.animalreferencepathology.com/



Advancing the art and science of veterinary medicine



[cid:image001.jpg@01CF8F87.A0BD4830]

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[Histonet] RE: Pathology software

[Elizabeth Chlipala]

Richard

There is a company that designs barcoding software for smaller  histology labs 
it's called Cerebrum 

The contact information that I have on file is this:

Gregg Lahti
gr...@cerebrumcorp.com

http://cerebrumcorp.com/

Good Luck

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Cartun, Richard
Sent: Wednesday, March 04, 2015 3:56 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Pathology software

Can anyone recommend an inexpensive pathology software package for a small 
Dermatopathology laboratory?

Richard

Richard W. Cartun, MS, PhD
Director, Histology  Immunopathology
Director, Biospecimen Collection Programs Assistant Director, Anatomic 
Pathology Hartford Hospital
80 Seymour Street
Hartford, CT  06102
(860) 972-1596
(860) 545-2204 Fax


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RE: [Histonet] CD68 and CD204 for dogs/pigs

[Elizabeth Chlipala]

Jan

MAC387 is a marker that we have used successfully in canine and porcine, not 
entirely specific to macs but may work for your situation.

LIz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

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1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jan Shivers
Sent: Tuesday, February 24, 2015 3:59 PM
To: histonet
Subject: [Histonet] CD68 and CD204 for dogs/pigs

Can anyone recommend vendors for CD68 and CD204 that work on dogs and pigs (on 
FFPE tissue)?  I don't seem to find much information on species 
cross-reactivity online.

Thanks much in advance.

--
Jan Shivers
Senior Scientist
IHC/Histology Section Head
Pathology Teaching Program
Veterinary Diagnostic Laboratory
University of Minnesota
1333 Gortner Ave.
St. Paul, MN  55108
612-624-7297
shive...@umn.edu
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RE: [EXTERNAL] RE: [Histonet] Question regarding dehydration

[Elizabeth Chlipala]

Julio

4 to 5 hours a station is way to long for processing samples of this size, 
regardless of what type of tissue or species they are from.  Graduated alcohol 
dehydration is better.  I would process samples of that size (unless it was 
bone, fat or skin) for no longer than 45 - 60 minutes a solution for manual 
processing.  

Here is what a typical processing cycle would look like once the tissue has 
been adequately fixed.

50% alcohol  (you want to start here if you are working with small animal 
tissue such as mice and rats)
70% alcohol 
80% alcohol
95% alcohol
100% alcohol
100% alcohol
Xylene
Xylene
Paraffin
Paraffin
Paraffin

I hope this helps, you may be able to get sections by trimming into the block 
and then soaking them on wet ice for some time (possibly an hour or so).

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

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-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Julio Benavides
Sent: Friday, February 13, 2015 8:43 AM
To: histonet@lists.utsouthwestern.edu
Subject: Re: [EXTERNAL] RE: [Histonet] Question regarding dehydration

Thanks Ryan. Tissu sections where thin, something about 2 and 3 mm. I think 
they were well fixed (buffered formalin) nut the problem was the dehydration. 
to go from formalin straight into 100% ethanol looks a bit too drastic to me.

Thanks for your thoughts

Julio

On 13/02/2015 16:44, Roy, Ryan wrote:
 Its well documented in the literature that using graded alcohols in 
 processing is advantageous to prevent hardening of the tissue.

 How thick are the tissue section cut that are being processed? It is also 
 well documented that sections should avoid being cut thicker than 3-4mm as 
 this prevents the penetration the fixative as well as the other reagents.

 4 hours in each reagent seems excessive... ask other people too since I have 
 limited experience.



 -Original Message-
 From: histonet-boun...@lists.utsouthwestern.edu 
 [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Julio 
 Benavides
 Sent: Friday, February 13, 2015 10:19 AM
 To: histonet@lists.utsouthwestern.edu
 Subject: [EXTERNAL] RE: [Histonet] Question regarding dehydration

 Hi Ryan,

 thanks a lot for your thoughts. These blocks were processed elsewhere and 
 sent to us for the cutting and staining. Tissues were dehydrated in five 
 consecutive baths of ethanol 100%, 5 hours each (manual processing). Then, 
 they went to xilene (three baths, 4 hours each) and paraffin (two batch, 
 1h30´ each). Truth is that I have never seen such processing before. In our 
 lab, with automatic processing, we begin with 60% ethanol and go to 70, then 
 95 and then 100 before ethanol/xylene.

 I was wondering if anybody has used the 100% ethanol processing before and 
 which was the influence over tissues.

 Thanks

 Julio


  Forwarded Message 
 Subject:  RE: [EXTERNAL] [Histonet] Question regarding dehydration
 Date: Fri, 13 Feb 2015 09:06:47 -0500
 From: Roy, Ryan ryan@va.gov
 To:   'Julio Benavides' j.benavi...@eae.csic.es



 If the tissue is fatty it will blow apart on the water bath. If the H and E 
 staining is poor its probably the processing.

 What is the exact procedure for your process including time that the tissue 
 was in each reagent? I would include this in a message to all of histonet as 
 well as some may more knowledgeable than myself.

 Also, I would recommend using a 95% solution prior to the 100% alcohol (s) if 
 not a 70-80% and 95%.


 Ryan Roy HTL (ASCP)
 Manchester Veterans Affairs Medical Center Manchester New Hampshire

 Disclosure: The content of this email does not represent the views or 
 opinons of the VA





 -Original Message-
 From: histonet-boun...@lists.utsouthwestern.edu 
 [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Julio 
 Benavides
 Sent: Friday, February 13, 2015 4:26 AM
 To: histonet@lists.utsouthwestern.edu
 Subject: [EXTERNAL] [Histonet] Question regarding dehydration

 Hi there,

 we are having problems trying to cut some embedded samples (they crumble in 
 the bath and the few cuts we manage to get into HE are crap). These are 
 formalin fixed samples (bovine foetal and placenta samples) which went 
 straight from formlin into 100º ethanol for the dehydration before clearing. 
 I was wondering if such drastic dehydration step (no 60º, 70º or 90º ethanol 
 before the 100º) could have damage the tissue. Has anyone have a similar 
 issue before? do you think the samples are ruined for histology?

 Thanks a lot for your help

 Regards

 Julio
 Instituto de Ganaderia de Montaña
 Spain

 

RE: [Histonet] paraffin sectioning-dry tissue? - long response - image analysis related

[Elizabeth Chlipala]

Lucie

You are not overthinking this at all if you are utilizing your sections for any 
image analysis applications.  You need to be able to standardize as much of the 
histology process as possible.  There are so many other parameters that can 
cause section thickness to fluctuate.  Such as the sharpness of your knife, how 
fast you turn the hand wheel.  Blowing on the block is not acceptable in our 
lab that will create thicker sections since you are warming up the block.   
Great care is taken to standardize what we do, from soaking blocks to how we 
collect the sections to placement on the slide, and how often we move our knife 
blade.  We routinely soak all of our blocks but we keep in mind so many other 
factors when we are providing histology for image analysis.  It starts at the 
beginning with fixation, it all has to be standardized, our goal is to decrease 
the potential for variability.  That is on our minds at every step through the 
histology process.

The other thing to consider is how well the algorithm functions - you need to 
determine the limits of the algorithm and when it will stop functioning 
properly, which is usually due to staining issues (over or under staining or 
inconsistent staining), section thickness and overall section and stain quality 
which is so important.  As a part of algorithm validation we test for these 
parameters we want to understand where we lose functionality, accuracy and 
precision of the algorithm.  So we look at different section thicknesses and 
how that impacts analysis, we look at over and under staining parameters to see 
how that affects the algorithm, etc.  

Just my two cents.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Lucie Guernsey
Sent: Thursday, February 05, 2015 10:22 AM
To: James Watson
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] paraffin sectioning-dry tissue?

By adding water, ice, or warm humidity (through exhalations) to the mix, 
though, doesn't the block contract/expand? Wouldn't that change the ultimate 
thickness of the section? I've always wondered how much it affects things.

Could your sections be 1 um (or multiple um!) thicker/thinner that you expect? 
What happens if individual blocks contract/expand differently (due to the 
amount of time left soaking, how far into the block you've cut since you last 
soaked, etc.)? I feel like you couldn't properly compare quantifications across 
a study if the thickness of your tissue is an unknown variable.

Am I overthinking this?

Lucie Guernsey
UC San Diego
(858) 822-5797
lguern...@ucsd.edu


On Thu, Feb 5, 2015 at 7:35 AM, James Watson jwat...@gnf.org wrote:

 We use a 5% glycerin in denatured alcohol for our 100% alcohol on our 
 tissue processor for routine animal tissues, this reduces the over 
 dehydration of the animal tissue and greatly reduces the time required 
 to soak the blocks.  Warning, if processing fat or cell pellets do not 
 use this, we switch the containers to straight 100% reagent alcohol for them.
 For fat we have a longer processing schedule and for cell pellets we 
 have a short processing cycle.

 James Watson HT  ASCP
 GNF  Genomics Institute of the Novartis Research Foundation Scientific 
 Technical Leader II, Histology
 Tel858-332-4647
 Fax   858-812-1915
 jwat...@gnf.org

 -Original Message-
 From: histonet-boun...@lists.utsouthwestern.edu [mailto:
 histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Caroline 
 Miller
 Sent: Thursday, February 05, 2015 6:41 AM
 To: Geoff
 Cc: histonet@lists.utsouthwestern.edu
 Subject: Re: [Histonet] paraffin sectioning-dry tissue?

 Yes, exactly what Mike and Geoff said. All mouse tissue, especially 
 liver, can be really dry and needs a 'soak'. I have left them for an 
 hour before now but don't leave it for longer than 4 hours though 
 because it can start to swell and de-process!

 You will still only get a few non-chattery sections so be gentle. 
 Thinner sections also help too (3-4.5). Plus low um polishing after 
 you trim

 Good luck! It is weird at first but you will get used to it!

 Caroline

 Sent from my iPhone

  On Feb 5, 2015, at 6:29 AM, Geoff mcaul...@rwjms.rutgers.edu wrote:
 
  This is common with mouse and rat tissues, they get over-dried 
  with a
 typical processing schedule.
  Soaking the face of the block with a kimwipe wet with ice water for 
  60
 -120 seconds will enable you to cut 10 nice sections, maybe more.
 
  Geoff
 
  On 2/5/2015 6:23 AM, Emily Brown wrote:
  Hello all!
 
  I just started sectioning mouse liver in paraffin and 

RE: [Histonet] Cryosectioning undecalcified bone

[Elizabeth Chlipala]

Orla

There is an article in the Journal of Histotechnology from a while ago from 
John Tarpley that addressed methods for this.  I have a pdf of it and I will 
send in another e-mail.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Orla M Gallagher
Sent: Thursday, February 05, 2015 10:56 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Cryosectioning undecalcified bone

Dear Histonetters,

We would like to develop cryosectioning for undecalcified mouse and rat bones. 
Previous attempts to use the CryoJane system from Instrumedics with an old 
Bright cryostat and solid tungsten carbide blade a few years ago didn't result 
in successful reproducible sections  - marrow without bone or bone without 
marrow on the slide, in spite of various freezing and prep.
protocols used by my colleague. I suspect the old cryostat and blade weren't 
helping either.

Have you any recommendations on the best cryostat to use to do this? We'd like 
to also use the cryostat for standard soft tissue sectioning. I've seen Leica 
mentioned in a few papers relating to CryoJane.

There appears also to be a tape transfer method from Kawamoto's Section-Lab Co. 
Ltd. (i...@section-lab.jp)

Does anyone use this method or know whether the consumables are still available 
for purchase, as the website seems dormant?

Thanks for any advice,
Orla

--
**
Ms. Orla Gallagher
Bone Analysis Laboratory
Mellanby Centre for Bone Research
Department of Human Metabolism
D Floor Medical School
University of Sheffield
Beech Hill Road
Sheffield
S10 2RX
UK

Website: http://mellanbycentre.dept.shef.ac.uk

Tel: 0044114-2713337 (office)
  0044114-2713174 (lab)
E-Mail:o.m.gallag...@sheffield.ac.uk


*STOP*: Do you really need to print this e-mail?

*BE GREEN:* Keep it on the screen.

http://www.shef.ac.uk/faculty/medicine-dentistry-health/2.9274/emailwording

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[Histonet] RE: use of denatured alcohol

[Elizabeth Chlipala]

Joanna

We are a research lab and have been using denatured alcohol for years, since 
1998 we also recycle and have not seen any impact on our IHC staining.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

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1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Joanna Bartczak
Sent: Wednesday, February 04, 2015 2:25 PM
To: 'histonet@lists.utsouthwestern.edu'
Subject: [Histonet] use of denatured alcohol

Hello,

Our lab is considering switching to denatured alcohol as a cost saving 
initiative.
Is  anyone using denatured alcohols and performing subsequent Class II IHC 
testing with no impact to results?

Thank-you,
Joanna

Joanna Bartczak
MLT II - Immunohistochemistry
Calgary Laboratory Services
403-770-3695



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RE: [Histonet] Looking for the ideal temperature ranges and humidity levels - histology lab

[Elizabeth Chlipala]

Jim

Its based upon the equipment requirements from each operating manual, we use 
the most critical instrument in the room and what the temp and humidity 
requirements are for that piece of equipment, we find those are usually the 
most stringent and the requirements for the other equipment in the room usually 
fall in that range.  

Liz 

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

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1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Vickroy, James
Sent: Friday, January 23, 2015 1:01 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Looking for the ideal temperature ranges and humidity 
levels - histology lab


We have always measured the temperature and humidity of the histology lab.   
Someone today asked what the normal ranges for a histology lab were?   Any 
ideas?

Jim Vickroy
Histology Manager
Springfield Clinic, Main Campus, East Building
1025 South 6th Street
Springfield, Illinois  62703
Office:  217-528-7541, Ext. 15121
Email:  jvick...@springfieldclinic.commailto:jvick...@springfieldclinic.com


This electronic message contains information from Springfield Clinic, LLP that 
may be confidential, privileged, and/or sensitive. This information is intended 
for the use of the individual(s) or entity(ies) named above. If you are not the 
intended recipient, be aware that disclosure, copying, distribution, or action 
taken on the contents of this information is strictly prohibited. If you have 
received this electronic message in error, please notify the sender 
immediately, by electronic mail, so that arrangements may be made for the 
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RE: [Histonet] taupathies, LB amyloid: IHC labeling followed by staining question

[Elizabeth Chlipala]

I find that when coupling IHC and special stains the best chromogen to use is 
DAB, unless the colors are too similar.  We would traditionally run the IHC 
first with the DAB chromogen which is not going anywhere and then essentially 
counterstain with whatever special stain you are performing.  Basically you run 
the special as you would normally after the IHC is completed.

I would not switch to a AP detection system most of those chromogens are not as 
permanent as DAB.  Brown IHC staining combined with the reddish orange from the 
congo red with a hematoxylin counterstain should look nice.  You can then 
dehydrate, clear  and mount in a permanent mounting media so why would you want 
to mount in aqueous.  I would keep it simple.

Good Luck.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Tyrone Genade
Sent: Friday, January 16, 2015 1:23 PM
To: histonet
Subject: [Histonet] taupathies, LB  amyloid: IHC labeling followed by staining 
question

Hello,

I found the following study, http://www.ncbi.nlm.nih.gov/pubmed/12000724,
where sections were probed with alpha-synuclein antibodies and then developed 
with DAB. Then the sections were stained with Congo Red for amyloid. The afore 
mentioned article references
http://jhc.sagepub.com/content/10/3/355 for the amyloid protocol. I assume 
they, the first article, had used the alkaline Congo Red method. The results 
look good and I can use this.

Does anyone have any experience in doing such a double labeling experiment?
Are there any problems that could be anticipated? If I use an alkaline 
phosphatase chromogen instead, could I expect problems? I am thinking of trying 
the MultiView kit by Enzo Life Sciences:
http://www.enzolifesciences.com/ADI-950-101/multiview-mouse-hrp-mouse-ap-ihc-kit/
and perhaps using some of the more vibrant chromogens:
http://www.enzolifesciences.com/platforms/immunohistochemistry/#An-Unrivaled-Selection-of-High-Definition-Chromogens
. Anyone have any references to papers that spell out a protocol? From what I 
can make out, the slides were first developed for IHC and then transferred to 
NaCl saturated 80% ethanol and then to the Congo Red where after the slides 
were dehydrated and cleared. (I assume, I could just as well mount in an 
aqueous mounting medium such as glycerol or
mowiol/n-propylgallate?)

Thanks for the answers to my previous question. Seems 10 micron is a good 
middle ground for the demonstration of the various pathologies from serial and 
double-stained sections.

Thanks
--
Tyrone Genade
Orange City, Iowa
tel: (+1) 712 230 4101
http://tgenade.freeshell.org

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[Histonet] RE: Paraffin Temperature Checks

[Elizabeth Chlipala]

All of our logs are use logs so we record when we use any piece of equipment 
and any QC or maintenance if required, we do not record or document anything if 
we do not use it, other than lab temperature and humidity which we record daily.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Cooper, Brian
Sent: Thursday, January 08, 2015 4:34 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Paraffin Temperature Checks

For those of you out there that are fortunate enough to have tissue processors 
or embedding centers that are used less often than daily-like maybe even weekly 
(or even less frequently,) how often are you checking and documenting the 
paraffin temperatures on said pieces of equipment?  For our daily equipment, 
it's no big deal obviously.  But we have a research processor and embedding 
center that doesn't get used often-sometimes for a week or two, and if we're 
not touching the machine, it seems overkill to document paraffin temps daily.

Thanks,

Brian D. Cooper, HT (ASCP)CM | Histology Supervisor Department of Pathology and 
Laboratory Medicine Children's Hospital Los Angeles
4650 Sunset Blvd MS#43- Los Angeles, CA 90027
Ph: 323.361.3357 Pager: 213-209-0184
bcoo...@chla.usc.edumailto:bcoo...@chla.usc.edu



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RE: [Histonet] Control strength? - personal opinion

[Elizabeth Chlipala]

Rene

I'm going to have to respectfully disagree with you.   In my opinion this is 
the one of biggest problems we have now a days when we  interpret our positive 
controls in histology.  We have been determining if staining is acceptable 
verses not acceptable for years as yes it stained no it did not stain and we 
really have to change how we look at our controls over time.  Staining 
intensity and consistency is key in determining overall quality of both special 
and IHC staining, especially when it comes to therapeutic markers and 
especially if we are utilizing image analysis for those markers like ER/PR and 
HER2.  We need to look at what we do over time and get away from what we have 
been doing in the past, such as acceptable verses not acceptable.  What we need 
to be able to define is the following especially in IHC staining - accuracy, 
precision, and robustness of staining just to name a few.   

It may be sometimes too late when a pathologist complains about it, that could 
mean that signal strength has been decreasing over time and has finally gotten 
to the point where the staining is inadequate.  I would think we would want to 
address trending problems sooner, since weaker staining intensity in control 
tissue could lead to false negatives in samples that do not express the protein 
we are trying to detect at lower expression levels.

This is what we do here.   We scan all of our positive IHC control slides that 
we utilize for image analysis and track staining intensity overtime either by 
visually looking at the positive control slides (many at a time) or running 
image analysis and then graphing our staining intensity in a Levy Jennings 
chart.  This is new for us and we are still working out the details on how we 
can interpret the data but we have found the information to be very valuable 
and enlightening.

Just my two cents for whatever it is worth.

Liz


Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Rene J Buesa
Sent: Monday, December 22, 2014 2:51 PM
To: Johnson, Carole; histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Control strength?

As I see it, control strength is less important than its ADEQUACY for 
diagnosis.Every time a pathologists signs a case requiring a special procedure 
WITH a control, it means that the control was adequate, UNLESS the pathologist 
complains about it.René J.  

 On Monday, December 22, 2014 4:00 PM, Johnson, Carole 
cjohn...@nmda.nmsu.edu wrote:
   

 Hello all,

How are you documenting trending signal strength of your controls for special 
stains and IHC? I was asked this question during an audit and would appreciate 
your help.

Carole Johnson
Carole Johnson, HT(ASCP)cm
New Mexico Department of Agriculture
Veterinary Diagnostic Services
505.838.9299

To understand is to stand under, which is to look up, which is a good way to 
understand




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[Histonet] RE: re: Flotation bath temperatures

[Elizabeth Chlipala]

Teri

We do, it's more of a combination use log for the microtome and tissue 
flotation bath, we record temperature and that we cleaned the unit, used fresh 
water and also cleaned the microtome.  This document also records the yearly 
calibration of the floatation bath and the yearly PM of the microtome.  I do 
need to add that both the calibration and PM will have additional documentation 
that is stored in a designated binder.  I can send you a copy of the form in a 
separate e-mail if you would like it.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Teri Johnson
Sent: Wednesday, December 03, 2014 12:04 PM
To: 'histonet@lists.utsouthwestern.edu'
Subject: [Histonet] re: Flotation bath temperatures

Dear all,

I am changing the language to flotation bath (instead of water bath, since that 
is entirely different lab equipment). I understand there is no longer a CAP 
requirement to record temperatures. Is there a GLP (or GxP) requirement to do 
so?

Teri Johnson, HT(ASCP)QIHC
Manager Clinical Trial Testing
Genoptix, Inc.
SAN5, Rm. 2005
760.516.5954 (office)
760.516.6201 (fax)




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subsidiaries. Any unauthorized review, use, disclosure or distribution is 
prohibited. If you are not the intended recipient, immediately contact the 
sender by e-mail and destroy all copies of the original message.
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RE: [Histonet] Reference to microtome micrometer thickness

[Elizabeth Chlipala]

Reuel

When your microtomes are serviced yearly they should be calibrated at the same 
time for micron thickness.   We have Leica microtomes and they are serviced by 
one of their vendors.  I had actually asked our service technician if they 
check for that and he said that do that, he also pointed out that your knife 
angle can also affect section thickness.  But that is just calibrating the 
microtome.  Overall section thickness can vary dependent upon many variables.

The type of disposable blade you use, how sharp that blade is, how many blocks 
you have sectioned with that particular blade,  how cold your block is, how 
fast or slow you turn the wheel of your microtome, some people blow on the 
blocks, which we all know makes sectioning easier, because it warms up the 
block and therefore you will get a thicker section, all of those parameters can 
affect your micron thickness.  Slight variation in section thickness may not 
impact routine HE slides for diagnosis, etc.  But when you are running IHC, or 
any stain and then utilizing image analysis that's where section thickness 
consistency is very important.

If a section is too thick you will be able to focus through multiple planes of 
nuclei in the sample, meaning you should see only a  single cell layer when 
tissue has been cut at the proper thickness.  

I hope this helps.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

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1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Reuel Cornelia
Sent: Friday, November 21, 2014 4:20 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Reference to microtome micrometer thickness

Hello Histonetters,
how will we verify the thickness of a paraffin section in a slide. Do we have a 
reference regarding  on how to measure the thickness. I based the  thickness of 
my section thru the mcirometer on the rotary microtome but one of our reveiwers 
does not believed that we are cutting them into
3 micron thickness or 4 micron thickness. Please if we have any reference, 
please share it to me. 
 
 
 
 
Reuel Cornelia, BS MT, AMT
Cellular Pathology
Texas Scottish Rite Hospital for Children
 Welborn Street
Dallas, TX 75219
Tel: 214-559-7766
fax: 214-559-7768

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[Histonet] RE: lab math: ihc dilution

[Elizabeth Chlipala]

Sheryl

Here is how I would approach the problem.  We would use the basic mathematical 
equation of the following  (in order for this to work you need to keep the 
units the same)

V1 x C1 = V2 x C2

V1 - volume of stock solution needed - this is what you are solving for
C1 - concentration of stock solution - you know this its 1.56 mg/ml or 1560 
ug/ml (there are 1000 ug in one mg)
V2 - volume of final solution needed - I put an arbitrary volume of 10 mls 
needed
C2 - concentration of final solution - you know this its 0.8ug/ml

V1 x 1560 ug/ml = 10ml x 0.8 ug/ml --- see how I made sure the units were the 
same I changed the mg/ml to ug/ml

V1 = .0005 mls  and then I would need 9.9995 mls of diluent to make up my 
0.8ug/ml concentration, you can change this to be expressed in microliters by 
multiplying each number by 1000.  

So in uls -  V1 = 0.5 uls and .5 uls

from there you can determine the titer which is your total volume divided by 
the volume of concentrated antibody

10/.0005

That would be a 1:20,000 dilution

For a dilution such as this I would make up a stock solution of either 1:100 or 
1:1000 and then dilute from there.

Now its time for the rest of you to check my math, did I do this correctly? 
 I went over it a few times, but you never know..

Have a GREAT Weekend!

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Stephenson, 
Sheryl
Sent: Friday, November 21, 2014 12:00 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] lab math: ihc dilution

To all you math whiz out there, please help with this math dilution.

If my Ab conc is 1.56 mg/mL and they want to optimize to 0.8 ug/mL, what 
dilution should I use?


Thanks,

Sheryl 




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[Histonet] RE: Recycler

[Elizabeth Chlipala]

The waste alcohol is disposed of with the rest of our waste chemicals in a 55 
gallon drum that picked is up by a licensed waste hauler.  The paraffin is also 
picked up.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Abbott, Tanya
Sent: Friday, November 21, 2014 12:25 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Recycler

We have a CBG Biotech recycler. We recycle Americlear, xylene substitute. How 
does everyone dispose of the waste alcohol and waste paraffin?
Thanks and have a great weekend!

Tanya G. Abbott RT (CSMLS)
Manager Technologist, Histology/Cytology St. Joseph Medical Center Reading, PA 
19603-0316 ph  610-378-2635 fax 610-898-5871
email: tanyaabb...@catholichealth.net

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[Histonet] RE: lab math: ihc dilution

[Elizabeth Chlipala]

Sheryl

I will add another bit of information.  That final antibody protein 
concentration is very low.  Most of our antibodies (around 60 to 70%) will be 
in the range of 1 to 10 ug/ml.  We do have some antibodies that can have some 
very low protein concentration though such as neurofilament.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Stephenson, 
Sheryl
Sent: Friday, November 21, 2014 12:00 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] lab math: ihc dilution

To all you math whiz out there, please help with this math dilution.

If my Ab conc is 1.56 mg/mL and they want to optimize to 0.8 ug/mL, what 
dilution should I use?


Thanks,

Sheryl 




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RE: [Histonet] RE: lab math: ihc dilution

[Elizabeth Chlipala]

Kathleen
I have a final volume of 10 mls not 1 ml in the equation I wrote.
Liz
Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.commailto:l...@premierlab.com
www.premierlab.comhttp://www.premierlab.com/

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

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1567 Skyway Drive, Unit E
Longmont, CO 80504

From: Kathleen Jones [mailto:kjo...@upei.ca]
Sent: Friday, November 21, 2014 12:50 PM
To: Sheryl Stephenson; histonet@lists.utsouthwestern.edu; Elizabeth Chlipala
Subject: [Histonet] RE: lab math: ihc dilution

Hi Liz,

Wouldn't you dilute in 999.5ul of diluent, not .5. 1ml=1000ul

Otherwise I got the same answer...

Kathleen

Kathleen Jones
Research Technologist
Pathology/Microbiology
AVC - UPEI
(902)213-2207


 Elizabeth Chlipala l...@premierlab.commailto:l...@premierlab.com 
 21/11/2014 3:35 PM 
Sheryl

Here is how I would approach the problem.  We would use the basic mathematical 
equation of the following  (in order for this to work you need to keep the 
units the same)

V1 x C1 = V2 x C2

V1 - volume of stock solution needed - this is what you are solving for
C1 - concentration of stock solution - you know this its 1.56 mg/ml or 1560 
ug/ml (there are 1000 ug in one mg)
V2 - volume of final solution needed - I put an arbitrary volume of 10 mls 
needed
C2 - concentration of final solution - you know this its 0.8ug/ml

V1 x 1560 ug/ml = 10ml x 0.8 ug/ml --- see how I made sure the units were the 
same I changed the mg/ml to ug/ml

V1 = .0005 mls  and then I would need 9.9995 mls of diluent to make up my 
0.8ug/ml concentration, you can change this to be expressed in microliters by 
multiplying each number by 1000.

So in uls -  V1 = 0.5 uls and .5 uls

from there you can determine the titer which is your total volume divided by 
the volume of concentrated antibody

10/.0005

That would be a 1:20,000 dilution

For a dilution such as this I would make up a stock solution of either 1:100 or 
1:1000 and then dilute from there.

Now its time for the rest of you to check my math, did I do this correctly? 
 I went over it a few times, but you never know..

Have a GREAT Weekend!

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.commailto:l...@premierlab.com
www.premierlab.comhttp://www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: 
histonet-boun...@lists.utsouthwestern.edumailto:histonet-boun...@lists.utsouthwestern.edu
 [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Stephenson, 
Sheryl
Sent: Friday, November 21, 2014 12:00 PM
To: histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu
Subject: [Histonet] lab math: ihc dilution

To all you math whiz out there, please help with this math dilution.

If my Ab conc is 1.56 mg/mL and they want to optimize to 0.8 ug/mL, what 
dilution should I use?


Thanks,

Sheryl




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RE: [Histonet] RE: lab math: ihc dilution

[Elizabeth Chlipala]

You are right I got it wrong, see no matter how many time you run the math, you 
still come up with problems

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: Truscott, Tom [mailto:ttrus...@vetmed.wsu.edu] 
Sent: Friday, November 21, 2014 1:01 PM
To: Kathleen Jones; Sheryl Stephenson; histonet@lists.utsouthwestern.edu; 
Elizabeth Chlipala
Subject: RE: [Histonet] RE: lab math: ihc dilution

When I solve your equation V1 X 1560 ug/ml = 10ml X 0.8 ug/ml I get .005 ml. 
Therefore the dilution would be closer to 1:2000. I would usually divide the 
1560 ug/ml by 0.8 ug/ml and get 1950 therefore 1:1950 would be more accurate . 
Tom T

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Kathleen Jones
Sent: Friday, November 21, 2014 11:50 AM
To: Sheryl Stephenson; histonet@lists.utsouthwestern.edu; Elizabeth Chlipala
Subject: [Histonet] RE: lab math: ihc dilution

Hi Liz, 
 
Wouldn't you dilute in 999.5ul of diluent, not .5. 1ml=1000ul
 
Otherwise I got the same answer...
 
Kathleen
 
Kathleen Jones
Research Technologist
Pathology/Microbiology
AVC - UPEI
(902)213-2207


 Elizabeth Chlipala l...@premierlab.com 21/11/2014 3:35 PM 
Sheryl

Here is how I would approach the problem.  We would use the basic mathematical 
equation of the following  (in order for this to work you need to keep the 
units the same)

V1 x C1 = V2 x C2

V1 - volume of stock solution needed - this is what you are solving for
C1 - concentration of stock solution - you know this its 1.56 mg/ml or
1560 ug/ml (there are 1000 ug in one mg)
V2 - volume of final solution needed - I put an arbitrary volume of 10 mls 
needed
C2 - concentration of final solution - you know this its 0.8ug/ml

V1 x 1560 ug/ml = 10ml x 0.8 ug/ml --- see how I made sure the units were the 
same I changed the mg/ml to ug/ml

V1 = .0005 mls  and then I would need 9.9995 mls of diluent to make up my 
0.8ug/ml concentration, you can change this to be expressed in microliters by 
multiplying each number by 1000.  

So in uls -  V1 = 0.5 uls and .5 uls

from there you can determine the titer which is your total volume divided by 
the volume of concentrated antibody

10/.0005

That would be a 1:20,000 dilution

For a dilution such as this I would make up a stock solution of either
1:100 or 1:1000 and then dilute from there.

Now its time for the rest of you to check my math, did I do this correctly? 
 I went over it a few times, but you never know..

Have a GREAT Weekend!

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Premier Laboratory, LLC PO Box 18592 
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Stephenson, 
Sheryl
Sent: Friday, November 21, 2014 12:00 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] lab math: ihc dilution

To all you math whiz out there, please help with this math dilution.

If my Ab conc is 1.56 mg/mL and they want to optimize to 0.8 ug/mL, what 
dilution should I use?


Thanks,

Sheryl 




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RE: [Histonet] HELP

[Elizabeth Chlipala]

Hans

We use the antibody from Serotec, it works quite nicely.  MCA711, proteinase K 
digestion,  rabbit anti-rat secondary and then Envision Rabbit (polymer).  We 
use this antibody at a 1:1200 dilution.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Hans B Snyder
Sent: Thursday, November 06, 2014 10:31 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] HELP

Hello All,

Does anyone have a protocol for the Anti-Neutrophil antibody [NIMP-R14]
(ab2557) for mouse tissue?  I have been trying to extinguish the background 
staining but cannot find the right combination.
​ We are doing this by hand. ​
 The recommended dilution for this antibody is 1:50 with HIER.   See used
protocol below.

So far I have tried:
1. concentrations 1:25, 1:50, 1:75, 1:100  - 1:50 gives very high background 
but good positive staining, 1:100 gives low background but not much positive 
staining.

2. HIER - 3, 5, 10,  20, minutes  @ 96C, 20 minutes at 60C  all give the same 
results - too much back ground

3. We have tried using the H2O2 before the primary and after, increasing the 
time from 30 minutes to 45 minutes.

4. Tried incubating in the primary for shorter time (20 minutes) and longer 
overnight at 4C.

5. Tried blocking using 3 different serums, first each one then combinations 
and longer times.

6. Tried cutting the DAB concentration in 1/2 then 1/3.


We have not tried enzyme or acid epitope retrieval yet.



*Does anyone have a working protocol or suggestions on what to try?*


​Thank you in advance.​





Procedure:

Deparaffinize

1.  Heat slides to 60C for 10 minutes (oven).

2.  Dry slides at room temp 5 minutes.

3.  Xylene 5 minutes.

4.  Xylene 5 minutes.

5.  100% ethanol 5 minutes (dehydration).

6.  100% ethanol 2 minutes (dehydration).

7.  95% ethanol for 2 minutes (rehydration).

8.  70% ethanol for 2 minutes (rehydration).

9.   Run in DI water for 10 minutes.



Antigen Retrieval

1.  Submerge in 0.01M Citrate Buffer (pH 6.0) at 96C for 5 minutes.

2.  Cold running water 5 minutes.



Staining

1.  Wash in PBS for 1 min.

2.  Permeabilize with 0.1% Triton X / 2.5% horse serum + 2.5% fetal
bovine serum for 2 hours.

3.  Incubate in primary antibody 30-45 minutes at in 2.5% horse serum
in PBS.

4.  Wash 3x in PBS/tween for 5 minutes each.

5.  Fresh 3.0% hydrogen peroxide (H2O2) 2x for 20 minutes each.

6.  Wash 3x PBS 2 minutes each.

7.  Incubate with anti-Rat Ig impress solution (according to vector’s
instructions) 45 minutes.

8.  Wash 3x in PBS for 5 minutes each.

9.  Prepare Impact DAB substrate (see insert).

10.  Using a light microscope, incubate sections with 50-100ul, until desired 
darkness occurs (5-30 seconds).

11.  Stop DAB reaction using deionized water.

12.  Wash in DH2O for 5 minutes

13.  Counterstain in Harris hematoxylin for 30 seconds.

14.  Wash in DH2O for 5 minutes.

15.  Dehydrate 2x in 95% alcohol for 30 seconds each.

16.  Dehydrate 3x in 100% alcohol for 2 minutes each.

17.  Clear 2x in xylene 5 minutes each.

18.  Mount coverslips using Leica mounting media.


Hans B Snyder
Histologistics
60 Prescott Street
Worcester, MA 01605
508-308-7800
h...@histologistics.com
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RE: [Histonet] Permanent red, Fuchsin and Vector Red not exactly red...

[Elizabeth Chlipala]

Yves

Try Biocares Warp Red, you will still need to air dry the slides prior to 
coverslipping do not place them in alcohol, air dry, place in xylene and then 
mount.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

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1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Yves Heremans
Sent: Thursday, November 06, 2014 1:12 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Permanent red, Fuchsin and Vector Red not exactly red...

Dear Histonetters,

I am trying to combine a peroxidase-DAB staining (brown) with an alkaline 
phosphatase-staining for detection of two proteins that do not overlap.
I have already tried Dako’s Permanent Red and Fuchsin+ and Vector’s Vector Red 
as AP substrates but I am not getting a clear red color, rather something 
brownish which is not very different in color from the DAB reaction product.
Is there anything I can do to obtain a clearer red color ?

Yves
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[Histonet] RE: Storage container for antibody vials

[Elizabeth Chlipala]

Richard

For the antibodies we store in the refrigerator we use the small plastic boxes 
from the pipette tips for the Dako antibodies.  For other vendors we use 
Eppendorf tube racks or the plastic freezer boxes that are around 5.5 square 
and contain 100 spaces.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

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-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Cartun, Richard
Sent: Friday, October 31, 2014 10:14 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Storage container for antibody vials

Can someone recommend a storage container for antibody vials that will fit 
nicely into a refrigerator?  Thank you.

Richard

Richard W. Cartun, MS, PhD
Director, Histology  Immunopathology
Director, Biospecimen Collection Programs Assistant Director, Anatomic 
Pathology Hartford Hospital
80 Seymour Street
Hartford, CT  06102
(860) 972-1596
(860) 545-2204 Fax


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[Histonet] IFN gamma

[Elizabeth Chlipala]

Happy Friday!!

I was wondering if the status of IHC staining for IFN gamma has changed since 
Chris Van Der Loos published the article back in 2001.  Back then he stated 
that it was not possible to stain for IFN gamma via IHC.

Immunohistochemical Detection of Interferon-gamma Fake or Fact?
Chris M. van der Loos,
Mischa A. Houtkamp,
Onno J. de Boer, Peter Teeling,
Allard C. van der Wal, and Anton E. Becker
Academic Medical Center, Department of Cardiovascular Pathology, Amsterdam, The 
Netherlands

Thanks

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.commailto:l...@premierlab.com
www.premierlab.comhttp://www.premierlab.com/

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[Histonet] RE: Antibody deposit visualization

[Elizabeth Chlipala]

This was years ago be we stained for the virus via IHC.  The antibody was 
supplied by the client.  That would seem to me to be the best method to 
determine deposits.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

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-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Rodriguez, 
Kristofer
Sent: Wednesday, October 22, 2014 9:31 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Antibody deposit visualization

One of my clients has been performing experiments with a mouse model of Dengue 
Virus Infection. They have been treating the mice with monoclonal antibodies 
(client did not specify what antibody)as a potential treatment for dengue virus 
infection. They have harvested the kidneys and put them in 10% NBF. They want 
to know if routine HE will show any antibody deposits.

I told him that I would not be able to directly observe the antibody deposit 
itself, however I might be able to observe the reaction to the antibody deposit 
in the basement membrane of the glomeruli as a membranous glomerulopathy on an 
HE.

I was wondering if I should do any special stains that might show more than an 
HE.
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RE: [Histonet] can you defrost and fix muscle tissue?

[Elizabeth Chlipala]

Tyrone

As long as the tissue has been frozen properly you should not have an issue 
with taking a frozen block to paraffin.  If the tissue has been frozen 
improperly (slow freezing) then you may encounter some freeze artifact.  We 
have done it on several occasions without any adverse effects.  We place the 
frozen sample directly into the fixative rather than thawing and then fixing, 
replace the fixative and then process as usual.  Sounds more like a processing 
problem to me, possibly over processed?  Once the blocks were trimmed did you 
place on wet ice prior to sectioning?  For fixation I would use 4% PFA or 10% 
NBF.  For staining I would use a modified trichrome one that combines elastic 
and trichrome staining since you may get disruption of the elastic fibers in 
atherosclerosis.  We did not work in rat models but we looked at rabbit and 
mouse models for atherosclerosis, we stained IHC for macrophages, smooth muscle 
actin and CD31.  Good Luck.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

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Longmont, CO 80504


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Tyrone Genade
Sent: Friday, October 10, 2014 1:44 PM
To: histonet
Subject: [Histonet] can you defrost and fix muscle tissue?

Hello,

A fellow faculty member had some rat muscle tissue that was set in tissue 
freezing medium for frozen sectioning. Some sections were cut (just as well as 
you may realize as you read on) and then the decisions was made to PFA fix and 
embed in wax. I didn't like the idea as it meant thawing the tissue and 
possibly introducing freeze-thaw artifacts and compromise the tissue...
But the decision was decided to proceed. The samples were incubated in 4% PFA 
for 24 hours. The pieces of tissue were about 1.5 to 2 cm (cubic). I took issue 
with the small volumes of 4% PFA were used (a student was set to do the task) 
and the volume was greatly increased and incubated for another
24 hours...

We then set about dehydrating, clearing and embedding in wax.

Today, with the student, I tried cutting a few ribbons but it was utterly 
hopeless. The sections kept crumbling as I cut (thin or thick sections).

Is the issue:
a) fixing frozen tissue
b) improper fixation
c) something else?

To exclude a problem with the wax, some sections of the wax (no tissue) were 
cut and they were perfect. Regretfully, no fresh muscle was fixed for 
comparison but that is going to happen if I am asked to repeat this.

For muscle tissue, would you in future suggest PFA or Bouin's? The person is 
interested in analyzing blood vessels. The rats are hypertensive so I'm 
thinking Bouin's in case a follow-up study, staining for collagen (scare 
tissue, artheroclerosis) is desired.

I would like to provide a good explanation for why the sections are crumbling...

Thanks
--
Tyrone Genade
Orange City, Iowa
tel: (+1) 712 230 4101
http://tgenade.freeshell.org

Romans 6:23: The gift of God is eternal life through Christ Jesus our Lord.
To find out how to receive this FREE gift visit http://www.alpha.org.
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RE: [Histonet] White Out:

[Elizabeth Chlipala]

It may not state it directly in either CAP or GLP guidelines but in a GLP 
compliant lab it is considered an industry standard not to use white out.  As a 
GLP compliant lab we have an entire SOP devoted to data entry.

If an error is made when data is being recorded or you notice an error at a 
later date when the form is being reviewed, even if it is on a QC checklist 
there are specific procedures that are followed.  You can not scribble over or 
abliterate what was written, you need to line through the error, make the 
correction and then explain why the form has been corrected, initial and date.  
We use a series of codes one of them being RE - recording error.  We have these 
codes posted all over the lab as a form so individuals will use the codes and 
record data properly.

I would have to say from experience that the biggest problem we encounter with 
data entry is scribbling over a recording error because that is a habit we all 
have and it does take some training to educate individuals in correct data 
entry.  

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 881-0763 cell
(303) 682-9060 fax
l...@premierlab.com

Ship to address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

From: histonet-boun...@lists.utsouthwestern.edu 
[histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jb 
[craiga...@gmail.com]
Sent: Tuesday, September 16, 2014 9:52 PM
To: Histonet@lists.utsouthwestern.edu
Subject: [Histonet] White Out:

 Histonet@lists.utsouthwestern.edu
Content-Transfer-Encoding: quoted-printable
Mime-Version: 1.0 (1.0)

Does anyone know where it says not to use white out on logs in the laborator=
y?  On logs, etc?

Thank you,

Sent from my iPhone=

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RE: [Histonet] Histotechnology certification for cytotechnologists

[Elizabeth Chlipala]

Bob

I believe this is correct, but others out there that may know more please 
correct me if I am incorrect.

If they have a BS they would fall in that category of eligibiity.  I would 
think most cytotechs do since they needed this to sit for the cytology exam.  
They need to have the proper college courses in science, etc.  The ASCP has 
this listed on their website then after one year of training and experinence in 
a histology lab they would be eligible to sit for the registry.

The route in BS or BA with one year on the job training.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 881-0763 cell
(303) 682-9060 fax
l...@premierlab.com

Ship to address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

From: histonet-boun...@lists.utsouthwestern.edu 
[histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Bob Richmond 
[rsrichm...@gmail.com]
Sent: Wednesday, September 17, 2014 7:08 AM
To: Histonet@lists.utsouthwestern.edu
Subject: [Histonet] Histotechnology certification for cytotechnologists

With declining utilization of gynecologic cytology (Pap smears)
opportunities for cytotechnologists are decreasing, and some of them are
considering adding histotechnology to their skills and certifications. One
small hospital's pathology service I'm acquainted with is trying to retain
its cytotechnologist by cross-training her as a histotechnologist.

Certification as a cytotechnologist requires all of the course work needed
for histotechnologist certification, so that isn't a problem. And
cytopreparation requires some of the needed skills for histotechnology -
though obviously not the primary skill of cutting paraffin. What are the
requirements for a cytotechnologist to sit the registry examination for a
histotechnologist? Is the National Society for Histotechnology (NSH)
considering addressing this issue?

Bob Richmond
Samurai Pathologist
Maryville TN
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RE: [Histonet] Neutrophil staining

[Elizabeth Chlipala]

Serotec has a rat anti-mouse neutrophil marker that works in FFPE tissues

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 881-0763 cell
(303) 682-9060 fax
l...@premierlab.com

Ship to address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

From: histonet-boun...@lists.utsouthwestern.edu 
[histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Hans B Snyder 
[h...@histologistics.com]
Sent: Saturday, September 06, 2014 3:43 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Neutrophil staining

Hello All,

I am looking to  stain neutrophils using special stains and antibodies
in mice tissue.  I wanted your opinion about the best methods to do
so.  I am thinking of using the may Grunwald giemsa and T-blue for
specials and myeloperoxidase and Ly6G for the antibodies.

What are your favorite special stains and antibodies to stain for neutrophils?

Best regards

Hans B Snyder
Histologistics
60 Prescott Street
Worcester, MA 01605
508-308-7800
h...@histologistics.com

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RE: [Histonet] detached retinas

[Elizabeth Chlipala]

Tyrone

I'm not sure if this will be helpful or not but we do a lot of work with mouse 
and rat eyes.  We have seen that with both 10% NBF and 4% PFA there will be 
retinal detachment regardless of how you process the samples.  Fixation in 
modified Davidsons or Davidsons in our experience is the only way that we are 
able to maintain good morphology of the retina without it detaching.  I'm not 
sure how that will affect the fixation of the entire fish, since I do not have 
experience in processing fish samples.  We have also found that you cannot 
process the eyes whole if you want any decent retina morphology, for us since 
we are just dealing with eye samples and not an entire fish, we will trim the 
edge off of both the rat or mouse eyes that we are running retinal studies on.

I hope this helps.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Tyrone Genade
Sent: Friday, August 29, 2014 11:12 AM
To: histonet
Subject: [Histonet] detached retinas

Hello,

I'm having a problem with detached retinas.

I fix my fish, whole, in 4% PFA for 24 hours. The belly is slit to allow better 
penetration of the PFA. The entire fish is about 4 cm long and about
6 mm wide. From the 4% PFA I proceed to decalsify the fish in 14% EDTA (pH 
7.6). This goes on for 3 days. From there the tissues is passaged through 70% 
to 95% to 100% ethanol and then cleared in methylbenzoate before being wax 
impregnated.

My current hypothesis is that the thick eye membrane is impeding the 
penetration of the PFA. As consequence, when I passage into the EDTA and 
alcohol I get rapid dehydration of the tissue and the change in pressure in the 
eye causes the detached retinas. How feasible is this hypothesis and what 
others can you come up with?

I am currently testing this hypothesis. I have fixed 2 fish in PFA but I have 
punctured the eyes of one of the fish to improve PFA penetration.

The sections I have cut of the first specimen show no issues with soft tissue 
fixation. My only problem is that the retina has detached formed several waves 
of tissue in the eye. The sections look good except for the eye issues. I never 
had this issue with Bouin's.

Thanks

--
Tyrone Genade

P.S. Incidentally, I am now using Paraplast Plus cat number 19216 ( 
http://www.emsdiasum.com/microscopy/products/histology/embedding.aspx ) and it 
is a dream to work with compared to what I was using. It is soft and cuts 
beautifully, the ribbons look good and impregnation is great (even by manual 
processing).

Orange City, Iowa
tel: (+1) 712 230 4101
http://tgenade.freeshell.org

Romans 6:23: The gift of God is eternal life through Christ Jesus our Lord.
To find out how to receive this FREE gift visit http://www.alpha.org.
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RE: [Histonet] whole mount immunofluorescence staining for mouse aorta

[Elizabeth Chlipala]

Mark

Not sure why they would want to do this for IF staining.  The only thing I can 
think of that is similar to this is in mouse models of atherosclerosis we have 
run en Face analysis of the mouse aorta.  The entire mouse aorta is dissected 
out, trimmed of fat and cut open via a specific procedure, the entire aorta is 
then stained with Sudan IV to demonstrate the plaques and then pinned on a tar 
plate, images are captured for image analysis.It is also common in the 
studies to run aortic root analysis - we have done this on frozen sections with 
brightfield IHC.  We have experience in all of these aspects of tissue 
collection and preparation, some of the techniques may apply to what they are 
trying to do.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Mark Elliott
Sent: Tuesday, August 19, 2014 3:48 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] whole mount immunofluorescence staining for mouse aorta

A colleague of mine has asked if I have any experience wholemount 
immunofluorescence staining for mouse aorta.  I don't have any experience with 
this so thought I would ask the group for any suggestions/protocols/ tips etc.
Thanks 
Mark

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RE: [Histonet] CD68

[Elizabeth Chlipala]

We use MAC387 not exactly the same as CD68 but stains macrophages, monocytes 
and I think granulocytes.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 881-0763 cell
(303) 682-9060 fax
l...@premierlab.com

Ship to address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
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From: histonet-boun...@lists.utsouthwestern.edu 
[histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Reuel Cornelia 
[reuel.corne...@tsrh.org]
Sent: Monday, August 18, 2014 8:27 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] CD68

Does anybody out there knows a CD68 antibody that works with
pig(Porcine) tissue. We have tried 3 CD68 antibodies already from Santa
Cruz, Dako and Novus that they say works with pig tissue but it did not.
We tried using heat antigen retrieval and enzyme digestion for paraffin,
it did not work. We also use pig frozen tissue and still do not work.
Any suggestion.





Reuel Cornelia, BS MT, AMT
Cellular Pathology
Texas Scottish Rite Hospital for Children
 Welborn Street
Dallas, TX 75219
Tel: 214-559-7766
fax: 214-559-7768
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RE: [Histonet] On the lighter side...

[Elizabeth Chlipala]

Too funny Richard - you would pass with flying colors

For me its 31 years - HTL-930

I have really been blessed, I love my job and I have really enjoyed my career.  
Every day there is something new to learn or to work on, for example the lab is 
putting the final touches on a poster that will be displayed at NSH this year, 
working on that has been exciting and a lot of fun too.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 881-0763 cell
(303) 682-9060 fax
l...@premierlab.com

Ship to address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

From: histonet-boun...@lists.utsouthwestern.edu 
[histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Cartun, Richard 
[richard.car...@hhchealth.org]
Sent: Friday, August 08, 2014 8:32 AM
To: Douglas Porter; histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] On the lighter side...

36 years; however, not registered.   Hopefully, I can take the QIHC someday 
and pass.

Richard

Richard W. Cartun, MS, PhD
Director, Histology  Immunopathology
Director, Biospecimen Collection Programs
Assistant Director, Anatomic Pathology
Hartford Hospital
80 Seymour Street
Hartford, CT  06102
(860) 972-1596 Office
(860) 545-2204 Fax
richard.car...@hhchealth.org


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Douglas Porter
Sent: Thursday, August 07, 2014 2:39 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] On the lighter side...

How long have you been a registered histotech?  36 years here.  You???



Douglas A. Porter, HT (ASCP)
Grossing Technician
IT Coordinator

Cancer Registrar


CAP-Lab, PLC
2508 South Cedar Street
Lansing, MI 48910-3138

517-372-5520 (phone)
517-372-5540 (fax)

 mailto:doug.por...@caplab.org doug.por...@caplab.org

 http://www.caplab.org/ www.caplab.org



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RE: [Histonet] Are Paraffin Blocks Biohazard

[Elizabeth Chlipala]

Dawn

I think it may be on the OSHA website under the bloodborne pathogen standard - 
fixed tissue is considered non-infectious and non-hazardous.  Below is what 
OSHA considers potentially infectious materials and specifically addressed 
unfixed tissue, fixed tissue would therefore be considered non-infectious.

occupational exposure to blood or other potentially infectious materials (OPIM) 
place workers at risk for infection with bloodborne pathogens. OSHA defines 
blood to mean human blood, human blood components, and products made from human 
blood. Other potentially infectious materials (OPIM) means: (1) The following 
human body fluids: semen, vaginal secretions, cerebrospinal fluid, synovial 
fluid, pleural fluid, pericardial fluid, peritoneal fluid, amniotic fluid, 
saliva in dental procedures, any body fluid that is visibly contaminated with 
blood, and all body fluids in situations where it is difficult or impossible to 
differentiate between body fluids; (2) Any unfixed tissue or organ (other than 
intact skin) from a human (living or dead); and (3) HIV-containing cell or 
tissue cultures, organ cultures, and HIV- or HBV-containing culture medium or 
other solutions; and blood, organs, or other tissues from experimental animals 
infected with HIV or HBV. The following references aid in recognizing workplace 
hazards associated with bloodborne pathogens.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Dawn Bugge
Sent: Friday, August 08, 2014 12:41 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Are Paraffin Blocks Biohazard

Hello Histo World!

Our pathologist for our private GI lab would like me to find out if anyone has 
done a study to determine if the paraffin blocks, once they have been 
processed, are considered biohazard.  I have searched high and low and can find 
many people stating that the blocks are not bioharzard, with the exception of 
neurological tissue, but they don't state how they know this.
He would like me to reference an actual study to prove that someone has 
actually looked into this.

Any one know of something like this?  I know common sense would say that once 
the tissues have been in formalin for hours, than on the processor for hours 
that the tissue would be non biohazard and completely safe.

Thanks for your help :)

--
Dawn R Bugge HT(ASCP), Lab Manager
Seattle Histology
Dawns Usborne Books Website http://x3128.myubam.com/ 
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[Histonet] RE: whole slide scanners

[Elizabeth Chlipala]

I agree with James the type of scanner you need will be dependent upon your use 
cases and workload.  We have an Aperio ScanScope XT which is an 120 slide 
scanner and that works well for us.

I'm going to make a shameless plug for a workshop that myself, Bill DeSalvo and 
Jesus Ellin will be giving at the annual meeting in Austin this year, it's 
called Digital Pathology for the Histotech - A Guide to Implementation it's an 
all day workshop that is on Saturday the 23rd and it will cover all aspects of 
how to select a scanner, how to implement scanning, etc.  It will be very 
valuable to histotechs that are already scanning and also techs that are new to 
the scanning process.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of James Watson
Sent: Tuesday, August 05, 2014 8:14 AM
To: 'Kalleberg, Kristopher'; histonet-requ...@lists.utsouthwestern.edu; 
histonet@lists.utsouthwestern.edu
Subject: [Histonet] RE: whole slide scanners

The scanner you get depends on your usage and volume.  Three that I recommend 
are for high volume.

Aperio, 
Good image quality,  if you need to link to study metadata Spectrum software is 
great.  Recommend for chromogen. Separate scanner for fluorescence,  
fluorescent scanner  only holds 5 slides at a time.  

Hamamatsu Nanozoomer,   
What we use,  we do high volume chromogen and fluorescence,  excellent for 
both.  Our NanoZoomer holds 210 slides for a chromogen run.  Can load up about 
100 fluorescent slides per night,  auto focus works well.  

Philips,
Chromogen only,  very fast scanning with high quality scans.  60 slides per 
hour.

There are many others on the market for smaller workloads.

James Watson HT  ASCP
GNF  Genomics Institute of the Novartis Research Foundation Scientific 
Technical Leader II, Histology Tel    858-332-4647 Fax   858-812-1915 
jwat...@gnf.org


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Kalleberg, 
Kristopher
Sent: Tuesday, August 05, 2014 5:18 AM
To: histonet-requ...@lists.utsouthwestern.edu; histonet@lists.utsouthwestern.edu
Subject: [Histonet] whole slide scanners

Hello All,

I am looking into the purchase of a whole slide scanner.  If anyone could 
supply some recommendations it would be greatly appreciated.  Thank you.

Kris
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RE: [Histonet] RE: whole slide scanners

[Elizabeth Chlipala]

We are a research based histology lab so we use scanning for several reasons:

1.  We scan slides for clients so they can review their images remotely and  
capture snapshot images, annotate, etc- study sizes may range from 10 to 100's 
of slides
2.  We scan all of our slides for IHC protocol development - our clients that 
request us to develop IHC staining protocols we scan slides so they can also 
view the slides and track our progress, plus this creates a great way for us to 
review the results too, we can easily determine the correct dilution by viewing 
multiple dilutions at the same time.
3.  We scan all of our stained slides from the control blocks and these are 
linked to our control block database so anyone can view the scanned slide to 
see if the controls are working properly.
4.  We have studies that require image analysis so all of the slides are 
scanned and then analyzed via a custom algorithm that will generate the data 
required for a particular study design.
5.  We scan all of our IHC control slides so we can tract staining consistency 

These are just a few of the applications that we use WSI for.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: Gudrun Lang [mailto:gu.l...@gmx.at] 
Sent: Tuesday, August 05, 2014 11:13 AM
To: Elizabeth Chlipala
Cc: histonet@lists.utsouthwestern.edu
Subject: AW: [Histonet] RE: whole slide scanners

Hi,
for which purpose do you use the high troughput scanner. Archive or diagnostics?
Gudrun

-Ursprüngliche Nachricht-
Von: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] Im Auftrag von Elizabeth 
Chlipala
Gesendet: Dienstag, 05. August 2014 17:16
An: James Watson; 'Kalleberg, Kristopher'; 
histonet-requ...@lists.utsouthwestern.edu; histonet@lists.utsouthwestern.edu
Betreff: [Histonet] RE: whole slide scanners

I agree with James the type of scanner you need will be dependent upon your use 
cases and workload.  We have an Aperio ScanScope XT which is an 120 slide 
scanner and that works well for us.

I'm going to make a shameless plug for a workshop that myself, Bill DeSalvo and 
Jesus Ellin will be giving at the annual meeting in Austin this year, it's 
called Digital Pathology for the Histotech - A Guide to Implementation it's an 
all day workshop that is on Saturday the 23rd and it will cover all aspects of 
how to select a scanner, how to implement scanning, etc.  It will be very 
valuable to histotechs that are already scanning and also techs that are new to 
the scanning process.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Premier Laboratory, LLC PO Box
18592 Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of James Watson
Sent: Tuesday, August 05, 2014 8:14 AM
To: 'Kalleberg, Kristopher'; histonet-requ...@lists.utsouthwestern.edu;
histonet@lists.utsouthwestern.edu
Subject: [Histonet] RE: whole slide scanners

The scanner you get depends on your usage and volume.  Three that I recommend 
are for high volume.

Aperio, 
Good image quality,  if you need to link to study metadata Spectrum software is 
great.  Recommend for chromogen. Separate scanner for fluorescence, fluorescent 
scanner  only holds 5 slides at a time.  

Hamamatsu Nanozoomer,   
What we use,  we do high volume chromogen and fluorescence,  excellent for 
both.  Our NanoZoomer holds 210 slides for a chromogen run.  Can load up about 
100 fluorescent slides per night,  auto focus works well.  

Philips,
Chromogen only,  very fast scanning with high quality scans.  60 slides per 
hour.

There are many others on the market for smaller workloads.

James Watson HT  ASCP
GNF  Genomics Institute of the Novartis Research Foundation Scientific 
Technical Leader II, Histology Tel    858-332-4647 Fax   858-812-1915 
jwat...@gnf.org


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Kalleberg, 
Kristopher
Sent: Tuesday, August 05, 2014 5:18 AM
To: histonet-requ...@lists.utsouthwestern.edu;
histonet@lists.utsouthwestern.edu
Subject: [Histonet] whole slide scanners

Hello All,

I am looking into the purchase of a whole slide scanner.  If anyone could 
supply some recommendations it would be greatly appreciated.  Thank you.

Kris