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 
>  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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Re: [Histonet] Transport training for formalin fixed specimens

[Elizabeth Chlipala via Histonet]

Gareth

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

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

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

Liz

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

Ship to Address:

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


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

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

[Tony Henwood (SCHN) via Histonet]

Hi Gareth,
You will probably have to design your own Continuing Education Session.
I would recommend the following topics:
1.  Nature of formaldehyde - safety aspects, explanation of the MSDS.
2.  What formaldehyde is used for
3.  Appropriate packaging of pots containing specimens in formalin
4.  Spill containment, neutralisation, clean-up procedures and Notification 
requirements.

Probably only take 30 minutes or so.
Issue each attendee with a certificate and record attendees for CAP.

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-Original Message-
From: Gareth Davis via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, 30 June 2016 9:18 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Transport training for formalin fixed specimens

Okay, is there a training course for just transportion formalin fixed 
specimens.  CAP told me last year that anyone picking up specimens for us has 
to go through a training course.  The inspector seemed to be more concerned 
with the employees knowledge of formalin and possible spills.
So, does anyone know what course I am suppose to be using?
 I have looked on the CDC website, but all the courses I find refer to specific 
infectious disease.
Thanks in advance.
--
Gareth B. Davis, HT (ASCPcm), QIHC
Yuma Gastroenterology
Yuma, AZ 85364
928-248-5259
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information. If you are not the intended recipient, please delete it and notify 
the sender.

Views expressed in this message are those of the individual sender, and are not 
necessarily the views of NSW Health or any of its entities.


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[Histonet] Transport training for formalin fixed specimens

[Gareth Davis via Histonet]

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

[Nancy Schmitt via Histonet]

Hi All-
I have a couple things I am looking for assist on today:

1.   Are your HT's doing frozen section cutting?  Are they also doing the 
frozen section gross and inking?  Are they staining the slides?  We are 
interested in doing this and looking for how others do.

2.   Do your HT's gross smalls?  What does that include? How did you train 
them and can you share that information?  Is there a guide out there you would 
recommend?
Appreciate your input,
Nancy Schmitt MLT, HT(ASCP)
Pathology Support Services Manager
Dubuque, IA

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[Histonet] Short term temp in Texas

[Cheryl via Histonet]

Hi Histonetters --
I need a short term histologist (registered) for a three week stint in Texas.  
GOOD lab, I'd go do it but the schedule doesn't line up for me :( 
Everything paid-- please respond via email with your resume.  
Starts July 11--
Holler!
ad...@fullstaff.org
Cheryl Cheryl Kerry, HT(ASCP) 
Full Staff Inc.  
ad...@fullstaff.org 800.756.3309 Phone & Fax

https://www.facebook.com/TheHistologyCompany/
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[Histonet] ARC immunohistochemistry- "sticky sections" in DAB

[Michelle Chang via Histonet]

Hi Gabrielle,

Are you doing floating sections?  Did you cut the tissue recently?  Do you
notice your tissue is also "sticky"? If you do, there is a possibility that
the tissue may be infected by bacteria. Try washing the tissue with PBS or
TBS with 0.1% sodium azide a couple time. Sometimes it help.

Good Luck

Best,
Michelle
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[Histonet] Re #2: μm H staining, WHOOPS!!

[Gayle Callis via Histonet]

Sorry about hitting send too soon. 

 

Repeat of things to try: 

 

a.  Do not use regressive hematoxylin and eosin where hematoxylin
can be overly differentiated i.e. removed from too thin sections.  Use
progressive hematoxylin i.e. Gill II or Gill III type formulation. DO NOT
use acid alcohol differentiation with progressive hematoxylin.  Try staining
longer, i.e. 10 min in Gill III, and use acetic acid clarifier only 1 or 2
dips or skip clarifying solution entirely. 

b.  Never use acid alcohol differentiation even with your
hematoxylin

c.   Use progressive hematoxylin, and do not clarify or use acid
alcohol differentiation solution.  Wash well for 1 minute in running tap
water then blue. 

d.   Increase the thickness of sections to see if this satisfies the
post-docs.  Start with 3 μm and 4 μm but stain these sections with
progressive H  If you don't need 2 μm, then go to a more routine  4 μm or
5 μm thickness.   You need to explain to these post docs about too thin
sections do NOT have enough tissue/cell left to stain well enough. 

e.   Treat sections with FRESH MADE 1% periodic acid for 10 min,
rinse well and stain with progressive H  This periodic acid technique is
found in Sheehan and Hrapchak Theory and Practice of Histotechnology book.
PA treatment might improve the staining with your sections by making more
groups on DNA available to hematoxylin.   However, I didn't find it improved
my thin section staining as much as I wanted.   The sections were just too
thin. 

f.Try Eosin-phloxine mixture, start dehydration in a few quick
dips in 95%, then proceed to 100% alcohols.   Eosin-phloxine is available as
ready to use or make up in the lab.   Sheehan and Hrapchak is also a source
of this eosin formulation.  

 

Good luck

Gayle M. Callis HT/HTL/MT(ASCP)

GCallis Histology Service, LLC.  

 

 

 

   

 

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Re: [Histonet] 2 um sections H staining

[Gayle Callis via Histonet]

You wrote:  

When I cut at 2μm 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?

 

 

First is a question.   Why do they require a  2μm thick section in the first
place?I had a pathologist many years ago fall in love with these very
thin section for all tissues with the same complaint of pale staining.   It
was explained to him that this thickness was excellent for bone marrow and
renal biopsies but too thin for the majority of other tissues.  Simple, you
are slicing through cells much of the time and leaving only cell walls for
staining.  It there isn't enough thickness there, then hematoxylin doesn't
have enough tissue thickness to be "vibrant", and the same for th eosin.
The pathologist went back to the former routine 5μm thick sections.   Some
laboratories do use  4 μm routinely. 

 

Things to try: 

 

1.   If this thickness is required to see basement membranes or marrow
cells

a.  Do not use regressive hematoxylin and eosin, but rather progressive
hematoxylin i.e. Gill III type formulation, and do NOT use any
differentiation solution which can remove hematoxylin. 

b.  Increase the thickness of section by trying 3 μm and 4 μm but use
progressive H  If you don't need 2μm, then go to 4 or 5

c.   Treat sections with 1% periodic acid for 10 min, rinse and then
stain with progressive H  This technique is found in Sheehan and Hrapchak
book.   It might improve the staining with your sections. 

d.

 

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

[Rene J Buesa via Histonet]

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 
 wrote:
 

 When I cut at 2μm 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?
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Re: [Histonet] Testicular biopsy for infertility

[Teri Johnson via Histonet]

Dr. Rich Cartun 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.  Thank you."



I can understand keeping Zenker's out of the lab because of the mercury, but 
I'm at a loss why you cannot continue to use Bouins? You can buy the fixative 
fully prepared, or you can buy saturated aqueous picric acid from which you can 
prepare it in house. Doing it this way should keep the safety people calm since 
you are not storing "dry" chemical, which is the hazard. I suppose each state 
is different with regard to disposal, but I have always disposed Bouins as 
formaldehyde waste.



Having said that, I would look for potential alternatives that contain acetic 
acid (since both fixatives use it) and might also consider a Zinc Formalin as a 
substitute for the mercury. So I'm thinking AZF (Acetic Zinc Formalin) or 
modified Carnoy (minus chloroform) might give you the nuclear morphology that 
is needed although both would still require some adjustment by the pathologists 
in recognizing their particular artifacts and how they can use it to aid the 
interpretation.

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

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




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Re: [Histonet] Bouins for testicular biopsies

[Gayle Callis via Histonet]

Richard, 

 

You wrote:  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.
 
***
 
I don't think Bouin's is forbidden in laboratories and we certainly used it
routinely for the Masson Trichrome connective tissue stain.  The problem is
having stock Picric Acid in crystal form, now frowned upon by chemical
safety people and eliminated from shelves these days.   I had no problem
storing stock picric acid under a layer of water and keeping crystals from
outside edges of lid.Zenkers is obviously not used due to mercury
content.   
 
Bouin's is still used for Masson's Trichrome staining and can be purchased
ready-made from Sigma, Fisher and elsewhere.   The key would be to use it to
fix testicular biopsies, with no more than 72 hour fixation.   Be careful to
wipe any drips from around lids where picric acid crystals form, collect and
dispose of this fixative per your lab's regulations.   
 
There is a B-5 substitute, sold by BBC,  which may do the job just as well.
This B-5 substitute is known to work well for bone marrow biopsies where
good nuclear detail is important, and may be a good option.  If your techs
are using Bouins for Massons Trichrome connective tissue staining, you could
get a small container for the biopsy.   
 
 
Good luck
 
Gayle M. Callis 
HTL/HT/MT(ASCP)
GCallis Histology Service LLC  
 
 

 

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[Histonet] RELIA Histology Careers Bulletin - 6/29/2016 Sizzling new histology opportunities and Edible Firecrackers for your 4th of July celebration!

[Pam Barker via Histonet]

Hi Histonetters!

Have a safe and Happy July 4th Weekend.

I Have A Fun Idea to Share for Pool Parties and Cookouts…
Sprinkle Red Pop Rocks And Blue Sprinkles On Iced Sugar Cookies To Make
Edible Firecrackers!!
I guarantee you will be the hit of your Independence Day Celebration!
Do you have any fun ideas for the 4th to share?

I have some exciting opportunities that I want to pass along as well.  Maybe
they will look good to you or someone you know.

RELIA’S Hot Histology Opportunities!!
IHC Tech   
Modesto, CA -Use your IHC Skills!

Histotech  Tyler, TX state of the art lab!
Histotechnician Norfolk, VA $15K Sign-on bonus
Lead Histotech   Fayetteville, AR (run your own lab)
Histotechnician Nashville, TN
Asst. SupervisorRoswell, NM-Days!! 
Histology Tech   Birmingham, AL days, dermpath
Senior HTL  Flagstaff, AZ  -IHC/ISH
Histotech  Hammond, IN 
All of these jobs are full time permanent positions with some of the finest
facilities in the country.  
My clients offer excellent compensation and benefits and most offer
relocation assistance or sign- on bonuses.

For more information for you or a friend please contact me toll free at the
office at 866-607-3542 or on my cell call/text at 407-353-5070 or shoot me
an email: rel...@earthlink.net   

Have a Safe and Happy 4th of July!!

Thanks-Pam

Thanks-Pam

Right Place, Right Time, Right Move with RELIA!

Thank You!
 Pam M. Barker
 
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 
www.facebook.com/PamBarkerRELIA
www.linkedin.com/in/reliasolutions
www.twitter.com/pamatrelia 






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[Histonet] ARC immunohistochemistry- "sticky sections" in DAB

[Gabrielle Pollack via Histonet]

To whom it may concern,

I am a undergraduate researcher at Vassar College. I am performing
immunohistochemistry on mice using Arc primary, anti-mouse biotinylated IgG
(1:200),ABC (Vector labs) and DAB. So far we have gotten really great
staining, however the last immuno I ran, the sections, once in DAB were
sticking to the bottom of the wells. Does anyone have any idea as to why
this would happen? Thank you!

Best,
Gabrielle
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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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