Re: HELP! IHC background

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From:"Tim Morken" <>
Date:Thu, 29 Jul 1999 21:02:41 EDT
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It's pretty much impossible for an outsider to tell you what is wrong with 
your staining. There are too many variables.

It's best to approach this sort of problem analytically. First, use all 
fresh reagents to repeat the experiment and include other tissues and 
controls you generally have success with. If it works, then you know one of 
your previous reagents is bad. If not, you know it is something with the 
tissue processing. You may be lucky and find that the tissue in question 
stains badly and the others stain fine. That narrows things down quickly.

Whether it is a bad reagent or bad processing, you should take the time to 
figure out exactly what caused the problem. That means doing elimination 
experiments to isolate the problem. If you do this you will learn something. 
If you don't you are probably going to have the problem again.

Tim Morken, B.A., EMT(MSA), HTL(ASCP)
Infectious Disease Pathology
Centers for Disease Control
1600 Clifton Rd.
Atlanta, GA 30333


Phone: (404) 639-3964
FAX:  (404)639-3043

----Original Message Follows----
From: Marcia Bentz <>
Subject: HELP! IHC background
Date: Thu, 29 Jul 1999 15:51:20 -0400

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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