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-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Erickson,
Jamie E
Sent: Monday, December 09, 2013 12:06 PM
To: histonet@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.erick...@abbvie.com
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