HELP! IHC background

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From:Marcia Bentz <>
Date:Thu, 29 Jul 1999 15:51:20 -0400
Content-Type:text/plain; charset="us-ascii"

Hello all,
I'm having problems with background/inappropriate staining on frozen mouse
kidney. I'm trying to stain for TNF alpha and have done so in the past
using this identical protocol with success. I'm using ABC Elite,
Avidin/Biotin blocking, and DAB kits all from Vector. After finding
nonspecific staining (basically the whole section turned dark brown within
1 minute)I went back to all of Vector's troubleshooting tips. Even
extending the avidin/biotin block to 30 minutes each and adding neither
primary or secondary Ab the tissue section is stained. I've tried
everything I can think of and the Vector tech person couldn't offer
anything new. The one thing that is different from my former successes is
that instead of fresh frozen tissue, I left the tissue in 30% sucrose
overnight before embedding in OCT and freezing. Could this somehow be
interacting with the ABC?
Any suggestions would be greatly appreciated as I'm currently pulling my
hair out and will soon be bald!
Thanks in advance,

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