Re: [Histonet] CD34 for primate tissue

[Croix, Denise via Histonet]

Hi Cathy,

When I was doing work with NHP tissue (both flow cytometry and IHC) there
were several sources that I utilized for info on antibodies that were
cross-reactivity with various NHP species.  The NIH hosts a NHP reagent
resource website (nhpreagents.org) that shows which commercially available
antibodies cross react with non-human primate tissues.  They note that
there are discrepancies reported about cross-reactivity with the QBEND10
antibody for  rhesus and possibly cynos.  You will need to still check with
the vendors to confirm if the antibody has been tested in FFPE specimens as
that is not indicated on the site but it should give you a starting point
to work from.

I've had personal experience with cyno & rhesus T cells where individual
monkeys would not react with certain anti-human antibodies.  For example, I
had a couple of research animals that were negative for a CD3 antibody but
yet they stained with CD4 and CD8 antibodies.

Good luck!

Denise


-- 
[image: Powered by Sigstr]
*Denise
A Croix, Ph.D.*
Pathology Solutions Specialist - PD-L1 SP142 Assay
Roche Diagnostics Corporation
Tissue Diagnostics Division

972-207-7305
denise.cr...@roche.com
www.ventana.com

[image: Powered by Sigstr]


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Re: [Histonet] How to get your stains more vibrant when cutting at 2μm?

[Angela Lamberth via Histonet]

Thank you all very much for your suggestions. I'm going to play around with
a progressive H&E when I return from vacation next month. I do have
safranin on hand but will need to order some phloxine to experiment with. I
will probably need to order some additional supplies to make the
hematoxylin. I'm not sure yet if I'll start with Mayer's or Gill's or maybe
even Erlich's.

I should note that cutting at 2 isn't required, but it is desired once they
saw that I can. And since I can, I aim to please! :-)

In addition, I'm looking forward to trying the oven dry method before
coverslipping. A rapid dehydration isn't really possible since I'm working
with a xylene substitute (Pro-Par) and have been battling eosin carryover
but that is a whole different animal for another thread.

Thanks again! I'll report back in a month or 2.

On Wed, Jun 29, 2016 at 10:27 AM, Rene J Buesa  wrote:

>
> Angela:
> "Pale" results are the trade-off for great quality very thin "2 µm"
> sections but you can always improve intensity somewhat .
> 1- your "regressive" stain, if it is "modern Harris" has the inherent
> problem of lacking mercury chloride and it is little you can do about.
> Perhaps if you use "progressive Mayer" you will get better results. You
> will not have to differentiate (with the intrinsic "danger" of leaving the
> section too pale) and if used fresh Mayer's can be a good approach.
> 2- as to the counterstain perhaps you should add safranine to the eosin
> (20% safranine + 80% eosin) and will get a darker red.
> 3- try to dehydrate as quickly as possible or even better, wash the
> sections in distilled water and place them in an oven at 60ºC for 10
> minutes and coverslip as usual. You will eliminate any "color wash" due to
> the alcohols.
> If you've not enough "trust" on dry/oven dehydration, try with some
> sections as a test. You will like the method.
> René
>
>
> On Tuesday, June 28, 2016 5:53 PM, Angela Lamberth via Histonet <
> histonet@lists.utsouthwestern.edu> wrote:
>
>
> When I cut at 2μm my H&Es and special stains look pale. How can I get my
> stains to pop or am I stuck with pale looking stains when sectioning that
> thin?
>
> I run manual specials and a manual regressive H&E. For H&E I've tried
> increasing my time in hematoxylin (beyond the manufacturer recommendation),
> diluting my acid alcohol differentiation, and increased time in eosin but
> the slides still lack the vibrancy that many of the postdocs desire.
>
> I use Shandon instant hematoxylin and alcoholic eosin by Thermo. Everything
> else I prepare in house from scratch. Any recommendations?
> ___
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
>


-- 
Angela Lamberth
Histology Technician II
Histology Core Lab
La Jolla Institute for Allergy & Immunology
9420 Athena Circle
La Jolla, CA 92037
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Re: [Histonet] Bouins for testicular biopsies

[Bob Richmond via Histonet]

Richard W. Cartun, MS, PhD in Hartford CT asks:

>>What are people fixing testicular biopsies in to evaluate infertility? In
the past, I believe fixatives such as Zenker's and Bouin's were used for
this purpose since they enhance nuclear detail. Obviously, those fixatives
can no longer be used.<<

I don't think Bouin's fixative has been entirely banned, but it's a
nuisance - the picric acid stains almost anything it gets spilled on, and
there are the regulatory issues.

I've had acceptable results with strongly acid fixatives, mostly with
Davidson's fixative (3 parts water, 3 parts reagent alcohol, 2 parts 37%
formaldehyde, 1 part glacial acetic acid).

I'd want to try it out on a non-critical specimen - either orchiectomy
tissue or animal material - before inflicting it on a real patient. I
suppose a spay-neuter clinic could help.

Bob Richmond
Samurai Pathologist
Maryville TN
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[Histonet] Embedding Centers - HistoStar vs. TEC 5?

[Mary Faith Encarnacion via Histonet]

Hello Histonetters!

My lab is on the hunt for a new embedding center.  We've demo-ed the Leica
Arcadia, the Thermo HistoStar, and the Sakura TEC 5.  The votes are split
between the HistoStar and TEC 5.  I'd like to know anyone's thoughts on
these two instruments...pros, cons, your experiences.  Are there any more
that we should try out?

Thanks so much!  Can't wait to hear your thoughts.

Best,

Mary Faith Encarnacion, MHA, HTL(ASCP)QIHC

Supervisory Histopathology Technician

Pathology and Lab Medicine Service

VA Palo Alto Health Care System

Phone: (650) 493-5000 x67327 (office); x65086 (lab)
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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 have had 
>> no luck.  I am using the Bond RX polyme

Re: [Histonet] Testicular biopsies

[Terri Braud via Histonet]

I must be missing the obvious.  Why can one no longer use Bouins?  We still use 
Bouins.

Terri L. Braud, HT(ASCP)
   3. Testicular biopsy for infertility (Cartun, Richard)
Message: 3
Date: Tue, 28 Jun 2016 20:41:47 +
From: "Cartun, Richard" 
What are people fixing testicular biopsies in to evaluate infertility?  In the 
past, I believe fixatives such as Zenker's and Bouin's were used for this 
purpose since they enhance nuclear detail.  Obviously, those fixatives can no 
longer be used.  Thank you.
Richard
Richard W. Cartun, MS, PhD
Director, Histology & The Martin M. Berman, MD Immunopathology & Morphologic 
Proteomics Laboratory 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] CD34 for primate tissue

[Eddie Martin via Histonet]

Hi Cathy,

I haven't heard of the cross reactivity with monkey species using Novocastra 
CD34. However, novocastra's antibody is a monoclonal antibody. You may get 
better staining utilizing a polyclonal CD34 instead. Thermo-Fisher and Abcam 
provide a polyclonal CD34. If you reach out to them, they may be able to 
provide you with a sample size 0.1 microliter if they have them available. 

I hope his helps!

Best,
Eddie


Sent from my iPhone

> On Jun 30, 2016, at 9:06 AM, Cathy M. Mathis/Comparative Medicine 
>  wrote:
> 
> 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 have 
>>> had no luck.  I am using the Bond RX polymer system and I've tried all the 
>>> epitope retrievals from Leica.  Now I am try epitope retrieval offline; 
>>> pronase, trypsin, pressure cooker with high and low pH solutions.  Still no 
>>> signal.  Any help would be greatly appreciated.
>>> Cathy
>> 
> 
> 
> 

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[Histonet] IHC staining on tissues placed in Kerasoft

[Paula Overn via Histonet]

Hello all,

Our lab uses Kerasoft to soften our nails and nail bed tumors. We recently
had a case where every IHC stain was negative (including  internal  positive)
with the exception  of cytokeratin AE1/3. Has anyone else experienced this?
Does anyone have a better solution to soften nails when IHC staining might
be needed?

Thanks!


Paula Overn
651-254-9652

Email: overn...@umn.edu
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Re: [Histonet] CD34 for primate tissue

[Cathy M. Mathis/Comparative Medicine via Histonet]

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 have had 
>> no luck.  I am using the Bond RX polymer system and I've tried all the 
>> epitope retrievals from Leica.  Now I am try epitope retrieval offline; 
>> pronase, trypsin, pressure cooker with high and low pH solutions.  Still no 
>> signal.  Any help would be greatly appreciated.
>> Cathy
> 


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[Histonet] UNSUBSCRIBE

[Ellenburg, Deborah via Histonet]

Thank you

Deborah Ellenburg, HT (ASCP)
Clinical Lead
St. Francis Health System
One St. Francis Drive
Greenville, SC  29601
Office Phone: 864-255-1582
Cell Phone:  864-444-8291
FAX (864) 679-8963




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[Histonet] Trichrome

[Lauren Sweeney via Histonet]

Hello all,

I was wondering if anyone has any experience with doing a trichrome and leaving 
it to mordant in Bouin's for longer than 24 hours. Does this overstain and ruin 
the stain?

Thanks,
L


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