[Histonet] RE: Histonet Digest, Vol 81, Issue 1

Perry, Margaret Margaret.Perry <@t> sdstate.edu
Mon Aug 2 10:49:15 CDT 2010

What antibody are you trying to use?

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Subject: Histonet Digest, Vol 81, Issue 1

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Today's Topics:

   1. Help with Biocare polymer detection kit!! (Jennifer Campbell)


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>
	<5658CBDB9EAE6545ABE50D2563D81BF8181FF1 <@t> VDXSERVER01.vdxpathology.local>
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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


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