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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet