[Histonet] RE: Help with Biocare polymer detection kit!!

[Sally Price]

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" <jcampbell <@t> vdxpathology.com>
Subject: [Histonet] Help with Biocare polymer detection kit!!
To: histonet <@t> 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