[IHCRG] RE: [Histonet] Mouse GranzymeB

Tue Jan 28 13:26:25 CST 2014

Patsy and Amos

I can appreciate your comment but we have been using Dako's Envision+ Rabbit on mouse tissue for years now and have never seen any cross reactivity to mouse tissue, it's pretty much our go to detection system for rabbit antibodies (both monoclonal and polyclonal) on mouse tissue.  We have however seen cross reactivity to porcine with Dako's envision +mouse and other companies anti-mouse polymer reagents.   My suggestion if you have not already done this is to try to use some other retrieval methods such as pH6 or enzyme (proteinase K or pepsin or even no retrieval).  Tissue fixation is important along with time an temp of retrieval - under fixation or over retrieval or a combination of both can cause problems.  If we find we are getting some background we will run some more intense blocking - such as the following:

1. commercially available superblock - these can be competitive and may potentially cause decreased sensitivity so you will need to watch out on these
2.  Normal serum - we normally use a commercial serum free protein block but on occasions we will use up to a 20% normal serum block for 30 to 60 minutes, if you want to continue to use the envision+ rabbit your choice would be 20% normal goat serum
3. Saturated Casein - we'll make this up in house

All are worth a try, good luck

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
liz <@t> premierlab.com
www.premierlab.com

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-----Original Message-----
From: ihcrg <@t> googlegroups.com [mailto:ihcrg <@t> googlegroups.com] On Behalf Of pruegg <@t> ihctech.net
Sent: Tuesday, January 28, 2014 12:11 PM
To: 'Amos Brooks'; histonet <@t> lists.utsouthwestern.edu; 'ihcrg Group, (E-mail)'
Subject: [IHCRG] RE: [Histonet] Mouse GranzymeB

Amos u might have to get an anti rabbit link that has been mouse absorbed.
Dako's anti rab envision is made in a goat but it is not mouse absorbed.
Southern Biotec or Jackson Labs are really good places to get absorbed links.  Still no reliable cd4 and cd8 for ffpe mouse tissue as far as I know, I have tried many that claim they work but have not consistently worked in my hands, we still do that with frozen mouse tissue not aldehyde fixed.  Are u doing HIER with the EDTA ph9?  In my experience especially with the higher ph u can over retrieve and cause undesirable nuclear staining.

Patsy Ruegg, HT(ASCP)QIHC
Ruegg IHC Consulting
40864 E. Arkansas Ave
Bennett, CO 80102
H 303-644-4538
C 720-281-5406
pruegghm <@t> hotmail.com
rueggihcconsultingpr <@t> outlook.com

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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Amos Brooks
Sent: Friday, January 24, 2014 10:09 AM
To: histonet <@t> lists.utsouthwestern.edu; ihcrg Group, (E-mail)
Subject: [Histonet] Mouse GranzymeB

Ok Musketeers,
     I am trying to detect cytotoxic T-Cells in formalin fixed paraffin
embedded mouse liver. GranzymeB should do the trick. I have a rabbit anti
mouse (and human and rat) GranzymeB from abCam (ab53097). The spleen that I
ran as a control with it works fine. Nice T-cells and no signifigant
background staining. The liver on the others hand has some lymphocyte
staining, presumably cytotoxic T-cells as expected,  but it looks like every
hepatocyte nuclei are also picking it up.  Grrr,  right?
     If this antibody had been raised in mouse,  I wouldn't be surprised.
Also,  if the spleen looked similar, I wouldn't be surprised. Do any of you
have any ideas ab pi ut what this might be? Alternatively, does anyone have
any ideas about another antibody that would work here?  CD4 or CD8 would be
great here,  but its FFPE mouse,  so sadly that's out (more grumbling).
    For the completionists out there,  I used EDTA pH9,  peroxide block and
detected the antibody (diluted 1:800) with Envision (rabbit) and DAB from
Dako.

Any suggestions would be vastly appreciated,

Happy Friday,
Amos
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