[Histonet] metal enhanced DAB

[Tyrone Genade via Histonet]

Hello,

I'm using the Co/Ni enhanced DAB from Histological & Histochemical Methods
(2nd Ed). Does the solution normally turn a blue-gray after adding the
H2O2? I am getting a lot of back ground and precipitate on the sections.
The pH is 7.3.

I had used a similar method for Western Blotting that took 5 minutes for
the solution to change color. This is a near instantaneous color change.

Thanks

Tyrone
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Re: [Histonet] Davidson's fixative and IHC question

[Anatoli Gleiberman via Histonet]

My experience with small tissue samples (such as half of mouse brain or slice 
of mouse liver) fixed in Davidson or other alcoholic formalin fixatives is to 
fix it no more than overnight and transfer into 70% ethanol (almost forever). 
The majority of antigens will be preserved much better than in regular NBF.

Anatoli Gleiberman, PhD
Director of Histopathology
Buffalo Biolabs LLC,
73 High Street
Buffalo, NY, 14203

Phone:716-849-6810x354
e-mail:agleiber...@buffalobiolab.com



-Original Message-
From: Jay Lundgren via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Monday, February 22, 2016 2:53 PM
To: Judi Ford
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Davidson's fixative and IHC question

Today I learned what Davidson's fixative is!  Thanks!

Anyway, so it's a strong alcoholic formalin.  The answer to your question is; 
yes.  You need to be careful of overfixation, thus masking the epitope in 
question.

From Dako:

 " Of the many pre-analytical variables which affect IHC and ISH results, 
fixation is probably the most significant, impacting many other variables such 
as antigen retrieval and epitope binding. Unfortunately, to date, no single 
fixative has proven to be ideal for all targets and detection methods. However, 
it is generally more deleterious for tissue to be ‘underfixed’, rather than 
‘overfixed’."

Make sure not to leave your specimen in Davidson's for more than 24 hrs, and 
then in formalin for *less* than 72 hrs and you should be OK.  If staining is 
unsatisfactory you might have to tinker with your antigen retrieval protocol 
(longer) or primary Ab incubation time (longer).

  Sincerely,

Jay A. Lundgren, M.S., HTL (ASCP)

On Mon, Feb 22, 2016 at 11:45 AM, Judi Ford via Histonet < 
histonet@lists.utsouthwestern.edu> wrote:

> Hi Everyone. Hope you all had a really good weekend. Thanks for the 
> replies to my double ihc question.
>
> I do have another question; this one is from a friend of mine. Her 
> client wants to do ihc on rabbit eye tissue. The tissue will be fixed 
> in Davidson's fixative for 24 hours then in 10% NBF. Will this have 
> affect future ihc projects?
>
> Thanks,
> Judi
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Re: [Histonet] H Troubleshooting

[Teri Johnson via Histonet]

Hi Cassie,

I would strongly suspect your tap water. If you have a tap water rinse before 
hematoxylin, the pH or treatment additives carrying over could be weakening 
your stain. We fixed this problem previously by adding a bucket of DI water 
between the tap water wash and the hematoxylin, and that fixed it for us.

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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[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
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
 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.
 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.



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[Histonet] FW: Advice regards NaDH and SDH stains?

[Bustamante, Lin via Histonet]

Is anyone out there able to help this student? We don't perform, less analyze 
these stains.
Thank you very much.
Lin.

+++
NaDH and SDH stains
We are planning to perform magnetic resonance spectroscopy on the cranial 
sartorius and vastus lateralis muscles of control dogs and dogs with Golden 
Retriever Muscular Dystrophy to evaluate for differences in the concentration 
of various metabolites and then compare this with NaDH and SDH stains of biopsy 
samples from the same sites. However, I am having some difficulty determining 
how to compare them because I do not know how these stains are normally 
analyzed; do you employ a grading scale based on the severity of change?

I am trying to confirm which statistical analyses to apply to compare the 
spectroscopic findings with the histological staining characteristics and thus 
really need to figure out exactly how the stains will be interpreted! I have 
been reading the literature to see how NaDH / SDH staining characteristics 
correlate with muscular dystrophy but it is proving a struggle. I was hoping 
you might be able to recommend a reference text?

Thank you so much in advance. If there is someone else you would prefer I 
contact about this then please do let me know.

Thanks again,

Sarah


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[Histonet] Histotechs and Supervisors needed in Texas and Arkansas. Can you help?

[Pam Barker via Histonet]

Hi Histonetters!
How are you? I hope you are having a great week!
I was hoping you might be able to help me.  I am presently working with a
client in Dallas in need of a histotech and a client in Amarillo in need of
histology supervisor.
I am also working with a client in Little Rock, AR in need of histotechs and
a client in Fayetteville, AR in need of a lead tech.
The help I need from you is do you know anyone that might be interested in
hearing about either of these opportunities?  If so could you please forward
my e-mail to them?
These are full time day shift permanent positions.
 
If you are interested in either of these positions please contact me ASAP
toll free at 866-607-3542, cell/text 407-353-5070 or via email at
rel...@earthlink.net
 
If you are interested in positions in other areas of the U.S. please contact
me as well. I have clients nationwide.  I will keep your resume confidential
and I won’t release it to anyone without your permission.
 
Thanks-Pam
cell: 407-353-5070
email: rel...@earthlink.net
 
Right Place, Right Time, Right Move with RELIA!

Thanks-Pam
Pam Barker
President/Senior Recruiting Specialist-Histology
RELIA Solutions
Specialists in Allied Healthcare Recruiting
5703 Red Bug Lake Road #330
Winter Springs, FL 32708-4969
Phone: (407)657-2027
Cell: (407)353-5070
FAX: (407)678-2788
E-mail: rel...@earthlink.net 
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www.linkedin.com/in/reliasolutions
www.twitter.com/pamatrelia 






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[Histonet] Davidson's fixative and IHC question

[White, Lisa M. via Histonet]

We have had failure of IHC on CD-20 when fixed in Davidson's.  So it is
possible.  The only thing to do is validate all stains you will use with
the Davidson's.

 

Lisa White

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[Histonet] Glyco Kit

[Bustamante, Lin via Histonet]

If you do Plastic embedding from a Kit, could you please share your experience?
Please mention Kit name in your response.
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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Re: [Histonet] Davidson's fixative and IHC question

[Tyrone Genade via Histonet]

Hello,

I am using Davidson's fixative to fix whole fish and I am very happy with
the results. I had tried 4% PFA but had issues with collapsed eyes and
detached retinas... No such issues with Davidson's or Bouin's. In my
experience I get sharper nuclear staining by H and better declacification
with Davidson's. The integrity of the brain tissues is good with less
shrinkage than with Bouin's.

I fix whole fish over night and then move them to 70% ethanol.

I had issues with autofluoresence with Davidson's (no surprise there) but
it was easily remedied using Gayle Callis' glycine protocol (available
somewhere on histonet). Same story for Bouin's.

Some antigens are lost and seem irretrievable.  The anti-GFAP antibody,
GA5*, worked fine on PFA fixed fish but the epitope couldn't be retrieved
in Bouin's fixed fish. I'm not sure about Davidson's but using heat induced
citric acid antigen retrieval (pH 5, 80 min at 60 oC) has worked for
several epitopes.

* If memory serves, this is a phosphate-epitope so that could be something
to look out for.

I would advise testing several different fixatives and seeing which works
the best for your application. If you are working with soft tissue, such as
brain, a zinc fixative might be better for the preservation of delicate
epitopes: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1919503/ .

Bye

Tyrone
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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] H & E Troubleshooting

[Cassie P. Davis via Histonet]

"Another theory floating in the lab is because we bleach the stain buckets over 
the weekend maybe the bleach is saturating the bucket and slowly leaching out 
diluting the hematoxylin later in the day. (I would think this would be the 
case for all the slides not just the last few)"

I guess I need to give more details...the first few runs look "fine" per my 
supervisor. If we didn't rinse the buckets I would think the first few runs 
would look pale too...Leaching out bleach later in the day seems illogical to 
me. This is why I am leaning toward the xylene being saturated (but we didn't 
have half the cases that we normally do and it was fresh Monday morning when 
this occurred) Or the tap water which is hooked directly to our stainer and can 
vary from clean to yellow to tan within a day. I had this issue with the tap 
water when I was at another facility a few block from this one in the same 
city. I just need another voice of reason to show my supervisor.



Cassandra Davis
Histology Technician
Anatomical Pathology Laboratory
Saint Francis Healthcare
701 N. Clayton Street
Wilmington,DE 19805
Office:  302-575-8095
Email:  cda...@che-east.org
www.saintfrancishealthcare.org

From: Podawiltz, Thomas [tpodawi...@lrgh.org]
Sent: Tuesday, February 23, 2016 1:28 PM
To: Cassie P. Davis
Subject: RE: H & E Troubleshooting

Hi Cassandra,

Does the Eosin looked washed out. If so it may be your bluing. I had this issue 
a year or so ago and it turned out that the bluing was to strong and it was 
effecting the ph of the Eosin and giving it a washed out look. Since I had a 
gallon of the stuff, I just adjusted the time in bluing and my water rinse and 
the issue went away.

I will say I have also had the issue with bleached containers, but when that 
happened everything look light.

Tom


Tom Podawiltz HT (ASCP)
AP  Section Head
LRGHealthcare
603-524-3211 ext: 3220



-Original Message-
From: Cassie P. Davis via Histonet [mailto:histonet@lists.utsouthwestern.edu]
Sent: Tuesday, February 23, 2016 1:12 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] H & E Troubleshooting

Hi Histo folks,

 We are having a fun time troubleshooting our H because the problem 
in not consistent...



Yesterday we poured fresh xylene, Hematoxylin , Eosin & bumped our alcohol 
since our ordered had not arrived yet. One of our doctor said our hematoxylin & 
eosin was readable but too light(he got 6 out of the 36 cases), the other (who 
got the 30 cases)said it was good.



We had half the number of slides going through staining than we normally do.

We don't let the last alcohol before xylene get pink from eosin.

Had it been a Thursday-Friday I would be concerned the xylene needed to be 
changed.

The Define and Bluing were made fresh.



My theory is the tap water could be off or the xylene needs to be changed.

Another theory floating in the lab is because we bleach the stain buckets over 
the weekend maybe the bleach is saturating the bucket and slowly leaching out 
diluting the hematoxylin later in the day. (I would think this would be the 
case for all the slides not just the last few)


Cassandra Davis
Histology Technician
Anatomical Pathology Laboratory
Saint Francis Healthcare
701 N. Clayton Street
Wilmington,DE 19805
Office:  302-575-8095
Email:  cda...@che-east.org
www.saintfrancishealthcare.org


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[Histonet] Thromboplastin for cell blocks

[Victoria Baker via Histonet]

Hi

I am looking for Thromboplastin to make cell blocks.  I can't remember
who we got it from years ago.

Any information is much appreciated.

Vikki

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

[Sanders, Jeanine (CDC/OID/NCEZID) via Histonet]

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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[Histonet] Issues with tissues while doing IHC/IF

[Catherine Simonson via Histonet]

Greetings and Salutations oh wise and wonderful histology people.

I am trying to work up several antibodies for IF on 10 micron thick mouse
brains.  These tissues were perfused and fixed in 4% PFA prior to my
receiving them.  I treated them with sucrose prior to freezing.  The issue
I am having is that the tissue is falling off the slides during staining.
Using charged slides and performing (attempting to, anyhow) on the Ventana
Discovery.  Any suggestions would be most helpful.  Thanks in advance.
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[Histonet] Collodian bag for cell blocks

[Julio Benavides via Histonet]

Hi,

I have a question regarding the use of UCSF Collodion Bag Cell blocks.  
once you do the bag, you pour in the cells in, let´s say 10ml of  
formalin. Then, how do you proceed? Do you spin down the cells with  
the bag inside a plastic tube? do you take then put the formalin and  
cut the top of the bag into a cassette? do you close the bag with a  
thread with the cells and formalin?


Any help with this would be greatly appreciated,

Thanks a lot

julio



WILLIAM DESALVO via Histonet  escribió:

What method are you using? Making your own coated tubes, UCSF or MD  
Anderson. Are you purchasing coated tubes? Once the tube is coated,  
dried (using hood) and filled w dH2O, there should be no odor.  
Always work w/ collodion under the hood. Ethyl ether is main  
component. Once in cassette and processed, there should not be odor.


Sent from my Windows Phone

From: Baldwin, Kathy via Histonet
Sent: ‎1/‎4/‎2016 2:07 PM
To:  
'histonet@lists.utsouthwestern.edu'

Subject: [Histonet] Collodian bag for cell blocks

Hi All
Was wondering if anyone is using the Collodian bag for cell blocks.   
We just got it and have been using it 'works great' but all the  
techs are complaining of the smell.. Our ventilation has been looked  
at and has passed however the smell still lingers in the room.  Any  
suggestions??



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
Cell 812-887-3357





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Re: [Histonet] Davidson's fixative and IHC question

[Julio Benavides via Histonet]

Hi,

in my experience, Bouin´s fixative can sometimes give you problems  
with IHC. Both because it seriously interfere with some antigens (in  
my case, I was doing IHC for lentivirus and herpesvirus and more than  
24h of fixation would drastically reduce IHC labelling) but also  
because it gives you non-specific labelling (in may case, ovine  
neutrophils came up positive with that lentivurs Ac).


Julio



Robert Byrd via Histonet  escribió:


Bouin's solution is an excellent fixative for fetal brains, we were still
using it routinely when I trained 10 years ago but has (and already had in
most places) largely fallen out of use due to the explosive risk associated
with dried picric acid residue in drainpipes etc. It does work remarkably
well on soft and macerated fetal brains though. I use 10% NBF now out of
necessity but might be able to track down a Bouin's protocol if you are
interested.

On Tue, Feb 23, 2016 at 5:11 PM, Hawkins, Hal K. via Histonet <
histonet@lists.utsouthwestern.edu> wrote:



We briefly experimented with using a fixative for stillborn fetal brains
that was a mixture of equal volumes of 20% formalin and 95% ethanol.  The
gross preservation and firmness of these very soft brains was improved, and
the histology results were fine, but as one might predict, some antigens
were not preserved for immunohistochemistry.


From: Jeff Jurczak via Histonet [histonet@lists.utsouthwestern.edu]
Sent: Tuesday, February 23, 2016 3:45 PM
To: Bob Richmond
Cc: Histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Davidson's fixative and IHC question

Is this a good fixative for fetal brain? Anybody have any suggestions as
to protocols? Do any of the readers here do fetal (human) brain on a
regular basis?

- Original Message -

From: "Bob Richmond via Histonet" 
To: "Histonet@lists.utsouthwestern.edu" 
Sent: Tuesday, February 23, 2016 3:22:50 PM
Subject: Re: [Histonet] Davidson's fixative and IHC question

Several queries about the possible effect of Davidson's fixative on
immunohistochemistry.

I have a lot of experience with Davidson's fixative, but none with its
effect on IHC.

Davidson's fixative is easily prepared:

3 parts tap water
3 parts ethanol or reagent alcohol
2 parts 37% formalin (not neutral buffered formalin)
1 part glacial acetic acid

It is quite destructive to H & E staining if fixation is at all prolonged.
Tissue should be taken out of it in not more much more than 24 hours.

The history of Davidson's fixative is somewhat obscure - I have a good bit
of information on it. John Kiernan has said he'll work on ascertaining more
about its history if he ever has the time.

Bob Richmond
Samurai Pathologist
Maryville TN
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