RE: thanks but i've tried all that

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From:"margaret blount" <>
To:"Marcia Bentz" <>, "" <>
Date:Fri, 30 Jul 1999 16:05:28 +0100

I hven't been watching the replies so this is perhaps a repetition, but how
about increasing the concentration of your serum block? It really sounds a
bummer. Good luck.

-----Original Message-----
From:	Marcia Bentz []
Sent:	Friday, July 30, 1999 12:26 PM
Subject:	thanks but i've tried all that

Hi all,
Thanks for the suggestions. Problem is I've already done everything
everyone has suggested. At the moment I don't have access to any other
tissues. All the products are relatively new (exp. 3/200) but in
systematically eliminating things INCLUDING both primary and secondary
antibodies, the tissue is still staining. I have blocked for peroxidase,
which through testing I found wasn't necessary. 
Here's what I've done:
substrate alone= no staining
blocking serum, ABC, substrate= inappropriate staining
blocking serum,avidin/biotin block (both 15 min and 30 min)= inappropriate
blocking serum, avidin/biotin block, secondary with nml mouse serum in
addition to rabbit serum, ABC, substrate= inappropriate staining

I'm using frozen sections (mouse kidney) fixed 10 min with 4%
Pharmingen's rat anti-mouse TNF alpha, Vector avidin/biotin block, ABC
Elite rabbit anti-rat IgG secondary, and DAB

I want to again stress I'm gettting staining when omitting both primary and
secondary antibodies. I'm clueless....

Any more suggestions out there?

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