So I am staining tumors that were implanted as cells subdermally into mice. The cells are human. I am trying to do Caspase staining on these tumors. The primary is an anti-human rabbit polyclonal, and I am using a polymer (Biocare Mach3) in lieu of the secondary antibody. The background is through the roof!! Could the reason be that the tumor was grown in a mouse and is having cross reactivity somehow? What species antibody should I be using instead? All my mouse monoclonal antibodies work perfect on the tissue, it's this stupid rabbit polyclonal!! I am blocking endogenous enzymes (peroxidase etc., DAKO), avidin and biotin (just to see if that would help...it didn't), and protein block (it's literally an hour worth of blocking!!), developing with DAB (Dako) and hematoxylin counterstain. I am so confused as how to get this to work! Also, it isn't just this particular antibody it is any rabbit polyclonal I have tried. Could it be the polymer? It is the one that Biocare suggested? HELP!!
Thanks in advance =) Sarah Goebel, BA, HT(ASCP) Histotechnologist Mirna Therapeutics 2150 Woodward Street Suite 100 Austin, Texas 78744 (512)901-0900 ext. 6912 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet