Re: [Histonet] frozen section problem

[Manfre, Philip via Histonet]

Hi Dorianne,
You need to freeze your tissue faster.  Ideally, isopentane placed in a 
metal cup, that is that is then frozen in a dewar of liquid nitrogen, works 
best.  The isopentane, once frozen, is thawed a little with a metal rod to 
produce a small liquid pool and your tissue is placed in this for about one 
minute.  You need some equipment for this procedure, such as the metal cup that 
can sit inside a small, open dewar of liquid nitrogen.  Alternatively, you can 
freeze directly in liquid nitrogen, though you need to beware of the tissue 
fracturing due to the sudden and extreme temperature reduction.  Slower 
freezing of tissue (sitting on dry ice, etc.)  allows ice crystals to form in 
the tissue, creating the vacuoles you describe.
I hope this helps.
Phil.

Philip Manfre, BA, HT (ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP81-406
770 Sumneytown Pike
West Point, PA 19486
215-652-9750
philip_man...@merck.com




-Original Message-
From: Bonello Dorianne M at Health-MDH via Histonet 
 
Sent: Friday, July 16, 2021 11:25 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] frozen section problem

EXTERNAL EMAIL – Use caution with any links or file attachments.

Dear all,


We are experiencing freezing artifacts on our frozen sections. Basically, we 
are seeing cavity-like structures under the microscope, mostly elongated, 
especially when it's a frozen section on brain tissue. This is most probably 
happening due to ice crystal formation. We're not using cryospray, relying only 
on the cryobar boost function.


Does anyone has a solution to this problem please?


Regards,



Dorianne Bonello
Allied Health Practitioner (MLS)
Histology Laboratory - Pathology
Health-Mater Dei Hospital


[cid:image001.jpg@01D67184.63288530]


T +356 +356 25456434

E dorianne.m.bone...@gov.mt


Mater Dei Hospital, Triq id-Donaturi tad-Demm, l-Imsida, Malta MSD 2090 | Tel 
+356 2545  | 
https://deputyprimeminister.gov.mt/en/MDH/Pages/Home.aspx
 | https://www.facebook.com/materdeihospital/


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Re: [Histonet] Mallory-Azan stain

[Manfre, Philip via Histonet]

The Bielschowsky Silver Stain is a good stain for Purkinje cells, so perhaps 
fibers as well.
Phil.

Philip Manfre, BA, HT (ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP81-406
770 Sumneytown Pike
West Point, PA 19486
215-652-9750
philip_man...@merck.com




-Original Message-
From: Bob Richmond via Histonet  
Sent: Monday, September 9, 2019 1:24 PM
To: Histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Mallory-Azan stain

EXTERNAL EMAIL – Use caution with any links or file attachments.

Betsy Molinari HT, ASCP at the Texas Heart Institute in Houston asks:

>>I have a researcher that wants to stain Purkinje fibers and has 
>>requested
a Mallory-Azan stain. I have no experience with this stain. I have looked 
online for information but am reaching out to you for personal advice.<<

Well, I'm 80 years old and have never seen a Mallory-Heidenhain azan stain
- it was something you read about in old books when I was young. I have a fair 
collection of old books, and found a method in Ann Preece's venerable _A Manual 
for Histologic Technicians_, 3rd ed, 1972. I can scan it for you if nobody else 
sends you one.

The stain requires aniline ("aniline oil"), which I don't think you're allowed 
to handle any more - it's a significant hazmat.

Your researcher is probably using a very old reference, and needs to be brought 
into the 21st century.

Bob Richmond
Samurai Pathologist
Maryville TN
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Re: [Histonet] Sections falling off

[Manfre, Philip via Histonet]

You can use 60 degrees if they are not for immunohistochemistry (IHC).  If they 
are for IHC, you need to be more cautious with temperature.  Some antigens are 
adversely affected or destroyed by excessive heat.  Histochemistry should work 
fine heating the slides that much and that would probably aid adhesion.

Phil.

-Original Message-
From: Pablo Sánchez Quinteiro via Histonet 
[mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, March 22, 2017 2:29 PM
To: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Sections falling off

Thanks for your hint Philip,

They are stored at 37º overnight. I learnt the problem with the trapped 
water. I am very careful with it but they keep spoiling.

They seem transparent.

Could I try 60 minutes at 60º oven just before xylen?

Pablo


El 22/03/2017 a las 18:53, Manfre, Philip escribió:

>
> -Original Message-
> From: Pablo Sánchez Quinteiro via Histonet 
> [mailto:histonet@lists.utsouthwestern.edu]
> Sent: Wednesday, March 22, 2017 1:39 PM
> To: histonet@lists.utsouthwestern.edu
> Subject: [Histonet] Sections falling off
>
> Dear Histonetters,
>
> I am in trouble with my paraffin sections -samples fixed in Bouin's liquid.
>
> Pablo,
>   I think your sections are not completely dry, after placing them on 
> slides.  Silanized slides can trap water between the sections and the slide, 
> almost appearing like a bulge.  You may need to use the corner of a paper 
> towel to "poke" the paraffin section and drain off the excess water.  Make 
> sure the sections look dry before starting the staining procedure.  They 
> should be transparent (translucent), not opaque (milky white).
>   That's my guess
>
> Phil.
>
> Philip Manfre, HT (ASCP)
> Associate Principal Scientist
> Merck Research Laboratories
> WP81-406
> 770 Sumneytown Pike
> West Point, PA 19486
>
> 215-652-9750
> 215-993-0383 (fax)
> philip_man...@merck.com
>
>
>
>
> Just after the first steps: Two xylol baths and 100% ethanol most of
> them are falling off. Slides are silanised.
>
> I do not think it was an adhesion problem. What do you think about?
> Embedding, cuttibg?
>
> Thanks a lot in advance
>
> Pablo
>
>
> ---
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> de virus.
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>
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Re: [Histonet] Sections falling off

[Manfre, Philip via Histonet]

-Original Message-
From: Pablo Sánchez Quinteiro via Histonet 
[mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, March 22, 2017 1:39 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Sections falling off

Dear Histonetters,

I am in trouble with my paraffin sections -samples fixed in Bouin's liquid.

Pablo,
I think your sections are not completely dry, after placing them on 
slides.  Silanized slides can trap water between the sections and the slide, 
almost appearing like a bulge.  You may need to use the corner of a paper towel 
to "poke" the paraffin section and drain off the excess water.  Make sure the 
sections look dry before starting the staining procedure.  They should be 
transparent (translucent), not opaque (milky white).
That's my guess

Phil.

Philip Manfre, HT (ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP81-406
770 Sumneytown Pike
West Point, PA 19486

215-652-9750
215-993-0383 (fax)
philip_man...@merck.com




Just after the first steps: Two xylol baths and 100% ethanol most of 
them are falling off. Slides are silanised.

I do not think it was an adhesion problem. What do you think about? 
Embedding, cuttibg?

Thanks a lot in advance

Pablo


---
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virus.
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Re: [Histonet] Metabisulfite rinse for PAS reaction (potassium vs sodium)

[Manfre, Philip via Histonet]

I almost always have used SODIUM metabisulfite for any version of PAS.  I am 
also familiar with procedures that go straight to tap water for the 
pot-Schiff's rinsing, though that was from a job in my past.  I seem to 
remember that working fine.

Phil.

Philip Manfre, HT (ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP81-406
770 Sumneytown Pike
West Point, PA 19486

215-652-9750
215-993-0383 (fax)
philip_man...@merck.com



-Original Message-
From: Angela Lamberth via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Monday, March 06, 2017 12:58 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Metabisulfite rinse for PAS reaction (potassium vs sodium)

In Carson’s 3rd edition, the reducing rinse following Schiff for PAS is
0.55% potassium metabisulfite (pg 137-138). For PAS/AB the reducing rinse
is given as sodium metabisulfite (Carson pg 149).



My other texts (Sheehan/Hrapchak as well as Vacca) list the reducing rinse
as sodium metabisulfite which makes sense to me since the Schiff is made
with sodium metabisulfite.



Is this a printing error in the Carson book? I appreciate any light anybody
can shed on this.

-- 
Angela Lamberth
Histology Technician III
Histology Core Lab
La Jolla Institute for Allergy & Immunology
9420 Athena Circle
La Jolla, CA 92037
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Re: [Histonet] PAS Stain

[Manfre, Philip via Histonet]

We switched to alpha amylase since the diastase we were ordering was not 
working.  We use a 2% solution of alpha amylase for 40 minutes at room 
temperature and it has been working fine.

Phil.

Philip Manfre, B.A., HT (ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP45-251
PO Box 4
West Point, PA 19486

215-652-9750
215-993-0383 (fax)
philip_man...@merck.com



-Original Message-
From: Joanne Clark via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, May 04, 2016 4:03 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] PAS Stain

Hi Histonetters, I'm hoping someone can help me troubleshoot our PAS diastase 
method.  We have been digesting the tissue in 0.5% diastase of malt in a 60 
degree oven for 30 minutes, but do not see the glycogen being digested out.  I 
have tried alpha amylase and beta amylase also without any luck.  Does anyone 
have any suggestions to get the digestion to work

Joanne Clark, BAAS, HT(ASCP)CM
Director of Histology

P.   (575) 622-5600
C.   (575) 317-6403
F.   (575) 622-3720
TF. (800) 753-7284

pcnm.com




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Re: [Histonet] Automated IHC instrument

[Manfre, Philip via Histonet]

Valerie,
We have 4 DAKOs.  They are work-horses and are a very "open" platform.  
You can use all of your own reagents, nothing proprietary if you choose.  This 
makes them good for research and optimizing your own protocols.  They are also 
very consistent and reliable for conducting the same protocol repeatedly.  Much 
cheaper to own and operate than a Ventana.  This is not a slam on Ventana.
Ventana strainers are quite amazing but way more expensive than a DAKO. 
 The Ventana is partially open, or not open, depending on how you look at it.  
You can force it to use some of your own reagents but you always rely somewhat 
on Ventana reagents.  These stainers are mostly designed to run the same set of 
protocols over and over again, consistently (e.g. hospital).  They are also 
useful when used by technicians who want to "set it and forget it".  If someone 
doesn't really know IHC, and the troubleshooting involved sometimes, then this 
is the unit for you if you can afford it.  There is also the high pressure 
sales team that accompanies any interest in Ventana units.  Don't underestimate 
their tenacity, especially if you agree to have a trial machine placed in your 
lab for testing.  They will not want to remove it - an assumed sale, if you 
will.
I am not on the payroll for anyone but Merck, lest anyone think I am 
trying to influence a sale one way or another.  These are my personal 
experiences and opinions that I hope you will find useful.

Phil.

Philip Manfre, B.A., HT (ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP45-251
PO Box 4
West Point, PA 19486

215-652-9750
215-993-0383 (fax)
philip_man...@merck.com



-Original Message-
From: Murphy, Valerie via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, May 04, 2016 11:52 AM
To: 'histonet@lists.utsouthwestern.edu'
Subject: [Histonet] Automated IHC instrument

Hello Histonetters,
Our tissue core is interested in purchasing an IHC instrument. It would  be 
used for the more routine assays such as ER/PR, Her2, Ki67 etc.
Can anyone make a recommendation? Leica, Ventana, Dako?

Thank you,

Valerie Ratliff  B.Sc HTL(ASCP)
Research Assistant
Department of Oncology
Karmanos Cancer Institute
4100 John R
Detroit, MI 48201

Telephone: (313) 576 8282
Fax: (313) 576 8306
E-mail: 
murp...@karmanos.org

Better treatments. Better outcomes.


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Re: [Histonet] No More Blog Posts -- Over and Out!

[Manfre, Philip via Histonet]

Delete button broken, huh.  I suppose it was necessary to attack a colleague to 
feel puffed up about yourself.  Bully for you!

-Original Message-
From: Rene J Buesa via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Monday, May 02, 2016 2:23 PM
To: Lester Raff MD; 'histonet@lists.utsouthwestern.edu'
Subject: Re: [Histonet] No More Blog Posts -- Over and Out!

Thank you VERY MUCH!René 

On Monday, May 2, 2016 1:54 PM, Lester Raff MD via Histonet 
 wrote:
 

 To My Lab Colleagues:

As my intent has never been to sow discontent or rancor, I think it is for the 
best if I no longer post links to my blog, lab related or otherwise.  Of course 
the blogs go on, and if anyone is interested in being added to my mailing list 
for future notifications, just drop me a line at 
les.r...@post.com  The mailing list is never used for 
any purpose other than announcing a new blog post. Be sure to let me know you 
are from the Histonet list!

I will continue to participate in any histology/pathologist discussions here as 
I have for many years.

Cheers,

Lester J. Raff, MD MBA
UroPartners
Medical Director Of Laboratory
2225 Enterprise Dr. Suite 2511
Westchester, Il 60154
Tel: 708-486-0076
Fax: 708-492-0203

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Re: [Histonet] Question re: accessory piece for tissue flotation bath

[Manfre, Philip via Histonet]

With regards to the HistoOrientator, it does removes wrinkles from the sections 
fairly effectively.  That being said, I would not use it on any sections 
intended for immunohistochemistry.  The hot plate on this device gets very hot 
and would no doubt damage or affect the antigenicity of such tissues.  Also, 
some practice is required to effectively use it.  If you do not drain the 
slides somewhat first, the tissues will quickly move all over the slide as they 
heat.  They will move quite a bit, sometimes butting up against each other.  If 
you drain them too long, then the tissues start to dry onto the slide, wrinkles 
and all.  So, as I said, spend some time practicing with this device with 
practice tissue until you get the hang of it.  It is a good tool that is tricky 
to use at first.

Good luck!
Phil.

Philip Manfre, B.A., HT (ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP45-251
PO Box 4
West Point, PA 19486

215-652-9750
215-993-0383 (fax)
philip_man...@merck.com



-Original Message-
From: Sanders, Jeanine (CDC/OID/NCEZID) via Histonet 
[mailto:histonet@lists.utsouthwestern.edu] 
Sent: Wednesday, February 24, 2016 8:36 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Question re: accessory piece for tissue flotation bath

Morning!

Can anyone of you share the functionality of:

Flotation Work Station w/ 8" x 8" x 2 1/4" (Deep Dish), HistoOrientator and 
Dryer

Curious if the HistoOrientator actually removes wrinkles as the description 
states.

Thanks!

Jeanine H. Sanders, BS, HT (ASCP), QIHC
Centers for Disease Control and Prevention
1600 Clifton Rd., NE MS/G-32
Atlanta, GA 30329
j...@cdc.gov
404-639-3590

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Re: [Histonet] Nuclear Bubbling

[Manfre, Philip via Histonet]

Sort of a rude response to someone looking for help.

-Original Message-
From: Rene J Buesa via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, February 16, 2016 1:12 PM
To: Vickroy, James; histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Nuclear Bubbling

If I remember correctly, this issue has been discussed previously.The general 
consensus as to the cause of nuclear "bubbling" (in reality a lack of staining 
in the nuclear area) has been attributed to an incomplete section drying.After 
the section has be "fished" from the water bath, if the slide is not set to 
drain the underneath water before drying, the nuclear components are dissolved 
hence when the section is stained, there is nothing to stain → "nuclear 
bubbling".I think this has been previously stated so I really do not understand 
posting this same question again.I do not think that posting again the question 
a different answer is going to be received.rené 

On Tuesday, February 16, 2016 12:32 PM, "Vickroy, James via Histonet" 
 wrote:
 

 
Struggling to find an answer.  We do a lot of GI biopsies in our lab.  
Sometimes they look wonderful without any nuclear bubbling, other times the 
bubbling is pretty intense.  Since nuclear bubbling is often attributed to 
incomplete fixation we of course have investigated the fixation times.  I do 
not find that the problem is fixation.  In fact some of the biopsies end up 
fixing for 48 hrs before processing. (weekend).  There was a suggestion last 
week or so that there might be water trapped under the slides after cutting and 
before staining.  I really thought that this might be the issue however I'm not 
sure at this point.  Extra drying seems to help but sometimes slides side by 
side are so variable, one with bubbles and one without.  I also don't believe 
the problem is in the processing schedule since the problem has shown up on 
both a rapid and a normal schedule. (therefore longer dehydration, clearing, 
etc.)

I am wondering if anyone else has worked with this issue.  Here are my 
questions:


1.        Could it be something that is happening with the tissue before it 
gets to the lab?  Usually a delay if fixation  causes other artifacts but not 
bubbling.  Could it be heat from the GI procedure?

2.      We do use blue sponges for our biopsies.  I know some say get rid of 
the sponges but has anyone seen this problem caused by usage of sponges?

3.      What about the heat stage in our Prisma stainer?


I am really getting frustrated.  Pathologists never complain however I would 
rather all of the tissue did not have the "nuclear bubbling".  Again we only do 
biopsies so I really don't think the standard old " not enough time in 
formalin" is the issue.  I have even wondered about variables such as we use 
recycled formalin, recycled Clearite III.

Any suggestions?

Jim



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.com



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Re: [Histonet] Hematoxylin Precipitate

[Manfre, Philip via Histonet]

Well Tim,  
In the 31 years I have been in histology, I obviously have done all lab 
work with my eyes closed, never looking at the equipment or solutions I have 
been using.  I guess my stint in a high throughput contract lab taught me 
nothing nor did my 25 + years in big Pharma, notorious for hiring every hack 
who knows what a microscope slide is.  You suggested that I sit around getting 
published... I am published, BTW, but that accounts for 0.01% of my experiences 
in the lab, yes, in the lab processing, embedding, sectioning (quite good at 
that), cryosectioning, performing and troubleshooting IHC and histochemical 
stains.
Something I may have learned, and maybe you will when you catch up, is 
not assume you know what someone else's work experiences may have been or are.  
Histology occurs in many environments with differing equipment, reagents, 
processing styles, etc.  Some are better than others.  I guess our inadequate 
processing methods fall short, leaving far too much tissue on the slide where 
it belongs, and thus we don't have big tissue globs clogging everything, nor do 
we see precipitate with the hematoxylin used.  Traditional formulations of 
hematoxylin, sure, absolutely filter.  For anyone who benefits from filtering, 
why not continue.  No harm will come.  I was merely trying to advise someone 
who seemed to have an issue I thought I could help with.  Now I realize all 
issues should be relegated to Tim, the Charles Churukian of our times.  Look 
him up, if you're stumped by the reference.  Maybe I'll head for Wacky Tabacky 
land for a vacation.  It is quite beautiful there...

Phil.

-Original Message-
From: Tim H via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, September 22, 2015 5:04 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 hydrati

Re: [Histonet] Hematoxylin Precipitate

[Manfre, Philip via Histonet]

Wow, I agree with Liz.  There should not routinely be "so much tissue washing 
off".  There is a fundamental problem, if this is the case.

With regards to hematoxylin, have you tried Gill's Hematoxylin, 1, 2, or 3?  
These do not need filtering and do not produce a precipitate.

Phil.

Philip Manfre, B.A., HT (ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP45-251
PO Box 4
West Point, PA 19486

215-652-9750
215-993-0383 (fax)
philip_man...@merck.com




-Original Message-
From: Elizabeth Chlipala via Histonet 
[mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, September 22, 2015 1:55 PM
To: Tim H; 'histonet@lists.utsouthwestern.edu' 
(histonet@lists.utsouthwestern.edu)
Subject: Re: [Histonet] Hematoxylin Precipitate

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

[Manfre, Philip via Histonet]

Denatured alcohol is used all the time.  It is generally denatured so people 
don't drink it.  This stems back to a time when the histology lab also made for 
a great bar and grill.  :-) 

Phil.

-Original Message-
From: Alida Bailleul via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Tuesday, September 22, 2015 11:28 AM
To: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Hematoxylin Precipitate

Dear Histonetters,

There is some *Denatured Ethanol* bottles in the lab I just started working
in (CDA, Fisher brand). Can this denatured ethanol be used for histological
processing? I have never used this product, only absolute pure ethanol.
Please advise.

Thank you very much

All the best

Alida Bailleul
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[Histonet] Paraffin Effects on IHC

[Manfre, Philip]

Our lab is considering switching paraffin in our histology labs.  They are 
being evaluated with an emphasis on routine H&E staining.  The IHC lab has some 
concerns about the effects this may have on IHC staining.  We have noticed 
slight differences in staining intensity already, during some evaluation 
experiments.  Does anyone have opinions either way, positive or negative, on 
the following products?  Of particular emphasis are any effects on the staining 
intensity (which also could relate to the ease of deparaffinization of the 
sections), ability to stain evenly, or anything else you may feel is relevant.


1.   Fisher Scientific TissuePrep T-565

2.  Leica Paraplast

3.  ThermoScientific Histoplast

Thanks all for your expertise! It's great that we have such a global forum for 
our field.

Phil.

Philip Manfre, B.A., HT (ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP45-251
PO Box 4
West Point, PA 19486

215-652-9750
215-993-0383 (fax)
philip_man...@merck.com


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[Histonet] RE: Oil Red O staining question

[Manfre, Philip]

We pretty much only used formalin-fixed, frozen tissue for ORO.  Just don't 
process them - the alcohol will dissolve the lipid out.

Phil.

Philip Manfre, B.A., HT (ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP45-251
PO Box 4
West Point, PA 19486

215-652-9750
215-993-0383 (fax)
philip_man...@merck.com





-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Elizabeth 
Chlipala
Sent: Thursday, March 20, 2014 12:30 PM
To: David Burk; histonet@lists.utsouthwestern.edu
Subject: [Histonet] RE: Oil Red O staining question

David

That does work, we have done it here on mouse muscle and liver,  but there are 
some problems to fixed and then frozen tissue.  The tissue  on occasion does 
not stay on the slide as well once it sectioned, even with plus 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

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 David Burk
Sent: Thursday, March 20, 2014 9:55 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Oil Red O staining question

Experts!

 

Is there any reason you would not consider formalin fixation followed by 
cryoprotection/cryosectioning for Oil Red O (or bodipy for that matter) 
staining of mouse muscle? 

I ask as many folks seem to have difficulty in the flash freezing aspect of 
tissue collection and end up with lots of ice crystal damage. While the above 
method is still subject to freezing artifact it does at least negate the 
'rushed collection' part of the process.

 

The literature favors flash frozen tissue but we've done successful staining of 
liver that has been fixed then cryoprotected prior to staining and things 
looked fine. Is there an issue with tissue adhering to slides after sectioning 
or some other reason this method is not preferred?

 

I value your opinion as I am wondering why one would choose one approach over 
the other.  Let's pretend tissue antigenicity isn't a factor - only section 
quality and lipid droplet staining.

 

Thanks for your comments!

 

David

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RE: [Histonet] Soaking artifact

[Manfre, Philip]

With respect, this may work in a non-industrial setting, but in Pharma, with 
high through-put, it is generally not possible to have individualized 
processing programs for a lot of different tissues.  You have to find one or 
two that work for the majority and compensate with some soaking.  Experience 
will help you know which tissues can't get left on too long.  Again, you can 
usually get past areas that look over-soaked just by cutting through them as 
you also mentioned. Just be careful, everyone, with small lesions so you don't 
ace through them.

Phil.
Philip Manfre, B.A., HT (ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP45-251
PO Box 4
West Point, PA 19486

215-652-9750
215-993-0383 (fax)
philip_man...@merck.com





-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Lee & Peggy Wenk
Sent: Monday, January 06, 2014 8:07 AM
To: Deanna Leslie; histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Soaking artifact

The only "soaking" artifacts that I can think of would be caused by:
- soaking too long in water (minutes instead of a few seconds)
- soaking under-processed tissue in water

In both cases, the tissue is supposed to be "protected" by the wax, and if 
it is not (under-processed), or if the faced block is in water too long, the 
tissue can start re-absorbing water. The tissue then turns white and swells 
out of the block. So all that "swelled out" tissue is cut away and lost when 
the tissue is put back on the microtomy for sectioning the ribbon.

If you soak for just a few seconds, such as a gauze with water being held 
against the block on the microtome, after it has been faced, then you will 
get a little bit of water absorbed into just a few layers of cells. Just 
enough to cut 2-4 sections. And you won't see that swelling artifact.

For those of you saying - "but I have to face all the blocks, put them back 
on ice and/or water while I cut a bunch more blocks, and then go back and 
cut each block" - that is an artifact also. You have over-dehydrated your 
tissue during processing, and you are putting back the water that you should 
not have taken out. Processing is supposed to remove the unbound water (not 
attached to proteins), and some of the bound water (attached to proteins), 
and leave some of the bound water (attached to proteins) in the tissue. If 
you HAVE to soak EVERY block for more than a couple of seconds, then you are 
wasting time rehydrating and wasting time while microtoming. Cut down the 
time in the alcohols on the tissue processor, to leave a little bound water 
in the tissues. And you can NOT processing little biopsies on the same long 
processing cycle as the larger pieces of tissue (uterus, breast, etc.). 
Those little biopsies will be over-dehydrated. They HAVE to be run on a 
separate cycle of much shorter time intervals (10-20 minutes in each 
solution (once fixed), instead of 45-60 minutes in each solution).

You should be able (on nearly every tissue block) to rough trim the tissue, 
and immediately start cutting ribbons. Possibly, you will need to put an ice 
cube on some of the harder tissue (cervix, uterus, bone, lens, etc.) just to 
get the paraffin hardness to match the hardness of the tissue. That being 
said, some tissues are naturally brittle or crumbly, and always need some 
water put back in the tissue, such as spleen or bloody tissue, but again, 
some wet gauze on the faced block for a few seconds should be enough time to 
get 2-4 sections. And that's all the tissue we usually need from those 
blocks. If you need more for IHC, put the wet gauze back on the faced block, 
and cut a few more sections.

Peggy A. Wenk, HTL(ASCP)SLS

-Original Message- 
From: Deanna Leslie
Sent: Sunday, January 05, 2014 4:42 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Soaking artifact

Has anybody in histoland ever heard of this?  I have been cutting tissue
for 25 yrs and until recently I had never heard of this!
I am under contract to a facility and the supervisor there does not want
anybody to soak their tissue or use ice!  Your are supposed to use the cold
plate, because as I have stated soaking them causing an artifact. I have
not disputed this because it is not my place or in my job discription as a
traveler.  I am not even sure what it is supposed to look like or what type
of problems it causes.

Thanks for listening!
Deanna Leslie HT ASCP
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RE: [Histonet] Soaking artifact

[Manfre, Philip]

I have been sectioning 28 years and, like everyone, completely disagree, at 
least with respect to animal tissue.  You can "oversoak" some tissues like 
brain and liver, but you can also cut through the over soaked region just by 
cranking the wheel a little under the puffy stuff is gone.  Also, the 
oversoaking occurs usually through neglect - leaving the blocks on wet ice for 
a long time.   My two cents.

Phil

Philip Manfre, B.A., HT (ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP45-251
PO Box 4
West Point, PA 19486

215-652-9750
215-993-0383 (fax)
philip_man...@merck.com




-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Deanna Leslie
Sent: Sunday, January 05, 2014 4:43 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Soaking artifact

Has anybody in histoland ever heard of this?  I have been cutting tissue
for 25 yrs and until recently I had never heard of this!
I am under contract to a facility and the supervisor there does not want
anybody to soak their tissue or use ice!  Your are supposed to use the cold
plate, because as I have stated soaking them causing an artifact. I have
not disputed this because it is not my place or in my job discription as a
traveler.  I am not even sure what it is supposed to look like or what type
of problems it causes.

Thanks for listening!
Deanna Leslie HT ASCP
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RE: [Histonet] Rolling of my ribbon ? Another paraffin question.

[Manfre, Philip]

Try running a wooden dowel (stick) lightly across your blade, dulling it 
slightly.  Disposable blades can actually be too sharp sometimes.


Philip Manfre, B.A., HT (ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP45-251
PO Box 4
West Point, PA 19486

215-652-9750
215-993-0383 (fax)
philip_man...@merck.com




-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Stella Mireles
Sent: Wednesday, November 13, 2013 3:18 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Rolling of my ribbon ? Another paraffin question.

*I am presently using a paraffin designated as an IM product.*
*We are a facility that cuts only autopsies and have been experiencing alot
of rolling of our sections.*
*We did recently switch to this product, because of cost.*

*Question :  Is the paraffin you are using working well on autopsy tissue
and producing ribbons right away ? Do you use it for infiltration as well
as embedding ?*

*Thank you for your assistant.*


*Stella Walters*
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[Histonet] RE: Inconsistent Sections with Cryostat

[Manfre, Philip]

I believe you may need to have the unit serviced.  It sounds like something is 
not tight enough, perhaps the stage or blade holder unit.  You said you secured 
everything which makes me think you have some issue with the cryostat itself.  
If a sharp blade, tightened blade and specimen, and varying the speed doesn't 
work, then you may need professional assistance form a service technician, 
especially if it is an older unit that has been moved around.  Have you tried 
different temperatures?  I don't think that is the problem but it is worth a 
try. 

Good luck! Cryosectioning can be an art in itself. Those decent sections are 
likely thicker than you intend, by the way.  Classic "thick and thin".


Philip Manfre, B.A., HT (ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP45-251
PO Box 4
West Point, PA 19486

215-652-9750
215-993-0383 (fax)
philip_man...@merck.com




-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Perow, Elliott 
S (PEROWES10)
Sent: Thursday, October 03, 2013 12:33 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Inconsistent Sections with Cryostat 

Hello All,

I have a Leica Cryocut 1800 with a Reichert-Jung 2020 microtome that has sat 
for 8 years and went through a move from one academic building to another.  I 
am trying to section brook trout brain, but am having difficulty getting 
consistent sections.
I will get a good section and then the next section will be a very very thin 
shaving of usually just the top portion of the OCT block.  I have tried 
adjusting the clearance angle, made sure the blade and specimen is secured, 
replaced the blade with a new one, have tried different sectioning speeds, 
different thicknesses of the section and still continually only get a decent 
section every other turn.  I was wondering if anyone has had any similar 
experiences or if maybe the machine just needs a maintenance check due to it 
being an older machine.  I wasn't sure if the advancement mechanism may be off 
or if it is another issue that doesn't seem apparent to me.  Any help would be 
greatly appreciated.

Thanks,

Elliott Perow
Juniata College
Biology POE


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RE: [Histonet] edge effect on IHC

[Manfre, Philip]

I concur with Rene'.  That was my first thought. Edges drying up on an 
autostainer due to hydrophobia or insufficient amounts of solution. Stainers 
usually have the option to adjust the volume that is applied and sometimes even 
the location on the slide (zones).  Thorough deparaffinization is also 
essential to avoid the hydrophobic repelling of solutions and wash buffers.  
Also be sure your reagents have some form of surfactant or detergent (e.g.Tween 
20) in them to break the surface tension and allow the even spread of whatever 
is being applied.

Phil.


Philip Manfre, B.A., HT (ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP45-251
PO Box 4
West Point, PA 19486

215-652-9750
215-993-0383 (fax)
philip_man...@merck.com




-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Rene J Buesa
Sent: Tuesday, September 24, 2013 10:36 AM
To: Mary Benoit; histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] edge effect on IHC

Sometimes that "edge effect" is caused by drying when there is not enough 
reagents. If you are using an automated stainer, please check the amount of 
delivered reagents.
René J.



From: Mary Benoit 
To: "histonet@lists.utsouthwestern.edu"  
Sent: Monday, September 23, 2013 5:00 PM
Subject: [Histonet] edge effect on IHC


I have been aware of some "edge effect" on an occasional slide, but lately we 
noticed the effect is showing up in the area of the slide that is farthest from 
the labels. We use DAKO Link Autostainer and their pretreatment baths for 
everything. One case in particular we stained a large melanoma met to colon 
that was well fixed and processed that stained only the edge, however when we 
sent block to Neogenomics reference lab, their slide stained evenly over the 
entire tumor area. We both used a melanoma cocktail. Not sure what is going on. 
Any suggestions?
Thank you
Mary F Benoit, MT(ASCP)
The Pathology Laboratory
830 Bayou Pines Drive
Lake Charles, LA 70601
mbp...@yahoo.com
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RE: [Histonet] (no subject)

[Manfre, Philip]

It's a bad question.  Generally the "best" temperature is probably between 55 
and 65, depending on the paraffin.  This would fall between the two latter 
answers.  Hmm  What to do?

Phil.

Philip Manfre, B.A., HT (ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP45-251
PO Box 4
West Point, PA 19486

215-652-9750
215-993-0383 (fax)
philip_man...@merck.com




-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Martin, Erin
Sent: Monday, September 09, 2013 9:28 AM
To: histonet
Cc: Naujokas, Agne; Meier, Melissa
Subject: [Histonet] (no subject)

Good morning all!

One of our fellows emailed me a question that she came across while studying 
for her boards:



"I'm studying for my board exam and came across questions re: paraffin 
embedding.
It reads: best temperature for paraffin embedding is
38-48
48-58
58-70.
I am getting some info on Internet that says 58 but is the range lower or 
higher than that? What do we use?"

This seems to me to be an odd question because it depends on the melting point 
of the paraffin in use.  Ours melts at 58C and we embed at 60C, but we have 
also used paraffin that melts at 56C and we embedded at 58C.  Or am I missing 
something?  Does anyone have a clear cut answer to this?



Thanks everyone!

Erin

Erin Martin, Histology Supervisor

UCSF  Dermatopathology Service
415-353-7248

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RE: [Histonet] Unsubscribe, Chapter 195

[Manfre, Philip]

Trained professionals should know by now that if you want to unsubscribe, you 
must type in all caps - UNSUBSCRIBE


Philip Manfre, B.A., HT (ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP45-251
PO Box 4
West Point, PA 19486

215-652-9750
215-993-0383 (fax)
philip_man...@merck.com




-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of 
nmhi...@comcast.net
Sent: Thursday, September 05, 2013 9:57 PM
To: HISTONET
Subject: [Histonet] Unsubscribe, Chapter 195

It is a concern that members of our  technically-oriented career field  have a 
difficult time understanding the method for unsubscribing to Histonet.  There 
is an  almost- daily posting to "unsubscribe", despite the fact that this 
subject has been addressed literally hundreds of times.  When one "joins" 
Histonet, instructions are provided, should be printed out for reference and 
used if the subscriber decides to leave the group.  We are required to be 
knowledgeable on all manner of technical routines requiring detailed 
instructions and  Histonet is no less clear in the methods for joining and 
"un-joining".  Use them, please.  Fire away - I'm retired and I can take the 
flak!  I do miss my microtome, though... 
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RE: [Histonet] Uncertified Histotechs

[Manfre, Philip]

I would look to the pharmaceutical industry, or industry in general, if they 
are hiring.  I believe they are more willing to hire without certification than 
a hospital.


Philip Manfre, B.A., HT (ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP45-251
PO Box 4
West Point, PA 19486



 

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jon Hannasch
Sent: Thursday, August 22, 2013 7:43 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Uncertified Histotechs

Is getting a job as an uncertified histotech a thing of the past? I have a 
friend who has been a very skilled histotech for many years and they have been 
looking for a job for about a year now. Is this due to bad interviewing or a 
lack of certification? I'm curious to see if this has happened to other people. 
They have applied at hospitals and bigger labs such as Caris. Im not asking for 
a job lead for them I'm just more curious if certification has become a 
prerequisite now.
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RE: [Histonet] Mixing Paraffin Brands

[Manfre, Philip]

Hey Pedro,
How are you?  We sometimes  use T-555, which is a little firmer.

Phil.

Philip Manfre, B.A., HT (ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP45-251
PO Box 4
West Point, PA 19486

215-652-9750
215-993-0383 (fax)
philip_man...@merck.com





-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Louro, Pedro
Sent: Wednesday, June 19, 2013 2:33 PM
To: Rene J Buesa; Lucie Guernsey; histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Mixing Paraffin Brands

Just out of curiosity, what paraffin would people out there recommend using for 
animal bone joints and turbinates?
We are currently using parapalst plus and was thinking of changing to a "harder 
paraffin"

Any thoughts???

Pedro L.


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Rene J Buesa
Sent: Wednesday, June 19, 2013 2:24 PM
To: Lucie Guernsey; histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Mixing Paraffin Brands

Each paraffin has some additives to improve either its penetration rate or 
density to section.
Mixing different melting points (MP) paraffin will result in another paraffin 
with an intermediate MP and the sectioning will be different.
Preparing the block with the mixture will probably cause so troubles while 
sectioning.
Why don't you just use them separate? There is no good reason to mix them and 
the two paraffins you mention are of good quality.
René J.

From: Lucie Guernsey 
To: histonet@lists.utsouthwestern.edu 
Sent: Wednesday, June 19, 2013 2:01 PM
Subject: [Histonet] Mixing Paraffin Brands


Hi,

Does anyone know if there's a reason why one shouldn't mix different brands
of paraffin? We normally use Fisherbrand's Paraplast Plus (melting point 56
C). We inherited about 8 bags of McCormick Paraplast X-tra (melting point
52 C). Will the different melting points be a problem?

If we were to use the McCormick paraffin, the only place it may mix with
the Fisherbrand paraffin is in the blocks themselves (as we refill the
embedder). But I don't want to compromise the quality of our blocks just to
not waste the free paraffin.

Or, another option could be that we use the McCormick in the processor and
the Fisherbrand in the embedder. Could that cause issues in the blocks as
the tissue would be infiltrated with one brand and embedded in another?

Maybe I'm over-thinking this..

Many thanks!
Lucie

Lucie Guernsey
UCSD
Dept. of Pathology
lguern...@ucsd.edu
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RE: [Histonet] Mixing Paraffin Brands

[Manfre, Philip]

Lucie,
Apparently I only responded to you directly.  I am re-sending my 
response in case anyone else on the net is interested.

Phil.


Lucie,

Personally, I would not mix them, as they have somewhat different properties, 
such as the melting point.   I think you are asking for trouble, or 
inconsistent results at least, when compared to your normal product.  If you 
have more than one processor and embedding center, set up one of each with the 
other paraffin and use it on low-priority tissue, if such exists.  At least the 
infiltration and embedding matrix will be the same. Just my opinion.  

Phil

Philip Manfre, B.A., HT (ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP45-251
PO Box 4
West Point, PA 19486

215-652-9750
215-993-0383 (fax)
philip_man...@merck.com



-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Lucie Guernsey
Sent: Wednesday, June 19, 2013 2:01 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Mixing Paraffin Brands

Hi,

Does anyone know if there's a reason why one shouldn't mix different brands
of paraffin? We normally use Fisherbrand's Paraplast Plus (melting point 56
C). We inherited about 8 bags of McCormick Paraplast X-tra (melting point
52 C). Will the different melting points be a problem?

If we were to use the McCormick paraffin, the only place it may mix with
the Fisherbrand paraffin is in the blocks themselves (as we refill the
embedder). But I don't want to compromise the quality of our blocks just to
not waste the free paraffin.

Or, another option could be that we use the McCormick in the processor and
the Fisherbrand in the embedder. Could that cause issues in the blocks as
the tissue would be infiltrated with one brand and embedded in another?

Maybe I'm over-thinking this..

Many thanks!
Lucie

Lucie Guernsey
UCSD
Dept. of Pathology
lguern...@ucsd.edu
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[Histonet] RE: What's your favorite mouse-on mouse detection kit

[Manfre, Philip]

Hey Brett,
We use Vector PK-2200.  Usually works well.

Phil.

Philip Manfre, B.A., HT (ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP45-251
PO Box 4
West Point, PA 19486

215-652-9750
215-993-0383 (fax)
philip_man...@merck.com




-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Connolly, Brett 
M
Sent: Tuesday, March 26, 2013 9:18 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] What's your favorite mouse-on mouse detection kit

Hi All,

For those of you working in research on animals tissue I'm looking for a 
consensus of opinions on kits for HRP mouse- on -mouse detection.

Would like to avoid biotinylation procedures (i.e.ARK).

Thanks,
Brett


Brett M. Connolly, Ph.D.
Principal Scientist, Imaging Dept.
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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[Histonet] RE: back up cryostat

[Manfre, Philip]

If you use anything often enough, it is wise to have a back-up, if possible. 
Microtomes, and especially cryostats, tend to malfunction without warning and 
can leave you "stranded". We have a back-up that isn't ours, but we have access 
to it.  That's another option.  Make friends with someone else who has one and 
strike an arrangement with them.

Philip Manfre, BA, HT(ASCP)
Associate Principal Scientist
Merck Research Laboratories
WP45-251
PO Box 4
West Point, PA 19486

215-652-9750
215-993-0383 (fax)
philip_man...@merck.com



-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of 
barbara.cr...@lpnt.net
Sent: Friday, September 21, 2012 8:07 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] back up cryostat

I would like to know what the consensus is on having a backup cryostat.
Does anyone have a back up cryostat?

I am nervous about not having a backup cryostat

Antoinette Crill
TEAM LEADER ANATOMIC PATHOLOGY
X5451



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RE: [Histonet] Please recommend xylene substitutes

[Manfre, Philip]

Jennifer,
Have you tried Histoclear?  It is great for deparaffinizing - we
still use xylene at the end of dehydration of slides but it might be
okay for that too.  We use xylene for processing.

Phil.
Philip Manfre, BA, HT(ASCP)
Senior Research Scientist
Merck Research Laboratories
WP45-251
PO Box 4
West Point, PA 19486

215-652-9750
215-993-0383 (fax)
philip_man...@merck.com



-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of jennifer
cresor mike hough
Sent: Tuesday, August 18, 2009 8:37 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Please recommend xylene substitutes

Hello All,

I am looking into switching to a different xylene substitute. I
currently use Slide Brite and am having problems with water in it.

Please recommend your favorite. Thank you for your response.

Jennifer
Temecula, CA
jenc...@ca.rr.com
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[Histonet] RA Lamb Lambwax Substitute

[Manfre, Philip]

Good morning Histonet,
Does anybody out there know of a good substitute for or have a
supply of RA Lamb's Lambwax paraffin?  RA Lamb was bought by Thermo and
Lambwax is no longer produced.  We used Lambwax in our lab for a
particular sectioning protocol.  It allowed for sectioning of small
tissue without removing, soaking, or chilling of the block.  This helped
keep the alignment of the block to the knife intact over a large number
of serial sections.
If anybody has experience with other paraffins that can offer
the qualities I described or know of a supplier that may have Lambwax,
please let me know.  T-565 and T-555 are not suitable for this specific
technique in our experience.
Thanks in advance,
Phil. 

Philip Manfre, BA, HT(ASCP)
Senior Research Scientist
Merck Research Laboratories
WP45-251
PO Box 4
West Point, PA 19486

215-652-9750
215-993-0383 (fax)
philip_man...@merck.com


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