Re: [Histonet] CD34 for primate tissue

[Cathy M. Mathis/Comparative Medicine via Histonet]

This is a great website for NHP info.  Thank you all for all your wonderful 
help!!
Hope everyone has a great 4th of July weekend
Cathy

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Sent: Friday, July 01, 2016 1:00 PM
To: histonet@lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 152, Issue 1

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Today's Topics:

   1. Re: How to get your stains more vibrant when cutting at 2?m?
  (Angela Lamberth)
   2. Re: CD34 for primate tissue (Croix, Denise)
   3. Just some before the long weekend Friday Fun-lab related  blog
  (Lester Raff MD)
   4. Certification (Adesupo, Adesuyi (Banjo))


--

Message: 1
Date: Thu, 30 Jun 2016 10:41:38 -0700
From: Angela Lamberth <alambe...@lji.org>
Cc: "histonet@lists.utsouthwestern.edu"
<histonet@lists.utsouthwestern.edu>
Subject: Re: [Histonet] How to get your stains more vibrant when
cutting at 2?m?
Message-ID:
<canunjwni6rswkgqvp5u7-wn3r29gk4mov_gr-ykwa41vtjt...@mail.gmail.com>
Content-Type: text/plain; charset=UTF-8

Thank you all very much for your suggestions. I'm going to play around with a 
progressive H 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 <rjbu...@yahoo.com> wrote:

>
> Angela:
> "Pale" results are the trade-off for great quality very thin "2 ?m"
> 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 60?C 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.
> Ren?
>
>
> On Tuesday, June 28, 2016 5:53 PM, Angela Lamberth via Histonet < 
> histonet@lists.utsouthwestern.edu> wrote:
>
>
> When I cut at 2?m my H 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 For H 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


--

Message: 2
Date: Thu, 30 Jun 2016 16:57:53 -0500

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]


[image: Powered by Sigstr]

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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 
> <cmmat...@wakehealth.edu> 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 
>> <cmmat...@wakehealth.edu> wrote:
>> 
>> Good morning my fellow Histo-netters, Does anyone have experience 
>> with any 

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 
> <cmmat...@wakehealth.edu> 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 
>> <cmmat...@wakehealth.edu> 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 
>>> <cmmat...@wakehealth.edu> 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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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 
> <cmmat...@wakehealth.edu> 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 
>> <cmmat...@wakehealth.edu> 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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Re: [Histonet] CD34 for primate tissue

[Eddie Martin via Histonet]

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 
> <cmmat...@wakehealth.edu> 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 
>> <cmmat...@wakehealth.edu> 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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Re: [Histonet] CD34 for primate tissue

[Cathy M. Mathis/Comparative Medicine via Histonet]

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 
> <cmmat...@wakehealth.edu> 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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Re: [Histonet] CD34 for primate tissue

[Eddie Martin via Histonet]

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] CD34 for primate tissue

[Cathy M. Mathis/Comparative Medicine via Histonet]

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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