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

[Montina Van Meter]

Jennifer,
I don't use the Mach 1 for animal tissue.  I am working with rat tissue
and use the Biocare Rabbit on Rodent or Mouse on Rat HRP or AP Polymers.
I have absolutely no background and my sections are 40um thick.  Perhaps
you are using the wrong kit.


Hope this helps,
Tina


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Perry,
Margaret
Sent: Monday, August 02, 2010 10:49 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] RE: Histonet Digest, Vol 81, Issue 1

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>
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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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