[Histonet] Caspase 3 on mouse spleen

[Elizabeth Chlipala]

Jackie

I have done quite a bit of work with cleaved caspase 3 on rat, mouse and
human tissue.  The antibody that I use is from cell signaling
technologies.  I have not performed the stain on mouse spleen, but I
have stained thymus, and mesenteric lymph node and I never have had a
problem with background staining.  In my hands this particular antibody
requires high pH retrieval and I do use dako's envision in my protocol.
I can provide you with a more detailed protocol and some images of
thymus and lymph node if you like.  But this is the basic protocol minus
rinses.

High pH retrieval
H2O2
Block
CC3 antibody 1:200 30 minutes RT
Envision + Rabbit 30 minutes RT
DAB +
Counterstain with Hematoxylin.

Hope this helps

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Histology Laboratory, LLC
P.O. Box 18592 
Boulder, Colorado 80308
Office: (303) 735-5001
Fax: (303) 735-3540
lizchlipala <@t> premierhistology.com
www.premierhistology.com
 
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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
Jackie.O'Connor <@t> abbott.com
Sent: Thursday, August 19, 2004 8:13 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Caspase 3 on mouse spleen

Good Morning Histonetters - 
Does anyone out there have experience with caspase-3 on mouse spleen?  I

routinely perform Caspase-3 staining on xenografts, some gut, and a
liver 
now and then - but just started looking at some spleens.  They're a
mess. 
I see what appears to be a lot of nonspecific staining of histiocytes in

the red pulp.  I'm staining immunocompetent mice, and repeated the stain

with a naive SCID spleen - same staining pattern.  Can anyone give me
any 
insight?  I'm using Pharmingen's 55965, Rabbit anti active Caspase-3.
I've 
tried DAKO Envision, as well as an SABC method and get exactly the same 
staining.  I'm a bit baffled.  Any tidbits?
Thanks!

Jackie O'


Jacqueline M. O'Connor HT(ASCP)
Abbott Laboratories
Global Pharmaceutical Research and Development
Discovery Chemotherapeutics
847.938.4919
Fax 847.938.3266
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