[Histonet] CD4 and CD8 mouse tumor IHC, thanks..

[gayle callis]

Another solution, and one we did almost exclusively for murine T and B cell
IHC was use biotinylated Rat anti mouse CD4 and CD8.   The isotype can be
purchased biotinylated from BD Bioscience/Invitrogen or you can buy Jacksons
Rat IgG-biotin.     

This totally eliminates a secondary antibody hence anything rat will not be
detected.   A bonus is staining is much faster too.  We routinely did
Streptavidin/biotin block from Vector since Streptavidin has an affinity for
integrins in epithelial cells.    Our normal serum block with biotinylated
rat antimouse primaries is 10% goat or donkey serum + 2.5% mouse.   The
block is also the diluent for the primary antibody and isotype/IgG negative
control.   Rabbit serum and antibodies were avoided since the bunny is
sticky, and can cause more background.   My immunologist frowned on anything
using a rabbit host unless we couldn't avoid it. 

Good luck
Gayle Callis
HTL/HT/MT(ASCP)  



-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Erickson,
Jamie E
Sent: Monday, December 09, 2013 12:06 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] CD4 and CD8 mouse tumor IHC, thanks..

Thank you to those that sent me suggestion on my CD4 problem. I have
recently discovered the problem.
The tumors are mouse tumors but they are injected in-vivo with a test
antibody that is Rat anti-mouse, specifically Isotype (Rat IgG2a). Hence
this is why my secondary Rabbit anti-rat stained so much..

Next I will try to block the Rat in-vivo injected antibody with a goat
anti-Rat secondary, as a  block prior to staining with CD4.
Or I could precomplex the CD4 with the Rabbit Anti-Rat and bind excess
Anti-Rat with Rat Serum.

Any thoughts or suggestion on this as always would be welcomed...

Thanks again..

Jamie

Abbvie Bioresearch Center
Pharmacology
100 Research Dr.
Worcester, Ma 01605
OFFICE    +1 508-688-3134
FAX          +1 508-793-4895
EMAIL  jamie.erickson <@t> abbvie.com
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