Jennifer: There are a variety possible causes for the background staining, which probably have more to do with the specimen you're attempting to stain than the detection systems, especially because you stated that you observed similar background with a biotin/avidin-based system. The first thing that comes to mind is whether or not Mach 1 is intended for use on canine/feline tissue; I suspect that its not, but you should check with Biocare's tech support to be sure. My guess is that either the immunologic enhancer reagent or antibody conjugated to the polymer-enzyme reagent is binding to endogenous immunolglobulins. Another possible cause of the background is that your tissue is over- retrieved/digested. It could also be that your antibody is not diluted adequately, or, as you suggested, the protein-blocking reagent is actually contributing to the problem. Good Luck! Sally ----------------------------------------------------------------------
Message: 1 Date: Sat, 31 Jul 2010 12:03:39 -0700 From: "Jennifer Campbell" <jcampb...@vdxpathology.com> Subject: [Histonet] Help with Biocare polymer detection kit!! To: histonet@lists.utsouthwestern.edu I have been trying to get the Mach 1 polymer kit from Biocare to work on my K9 and feline tissue and I am getting terrible nonspecific staining in my positive and negative controls. It appears to be sticking to something in the glands of the intestinal epithelia. I am also getting nonspecific staining in my LN tissue. I decided to switch over to polymer detection kit because I had been dealing with the issue of endogenous biotin when using the streptavidin biotin-based detection system, and now I am getting this other nonspecific staining! I have tried doing runs eliminating my protein block and eliminating the negative control and primary antibody step altogether (to see if maybe the protein block or the diluent used in my negative control and antibody dilutions are causing the problem). I have also reduced the incubation time of both the probe and polymer from 15 min and 30 min, respectively, to 10 min each. I have tried cutting down my antigen retrieval time substantially as well. I'm not sure what else could be causing this. Any suggestions?? Thanks in advance, Jennifer Campbell _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet