Re: Immunostaining (background)

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From:"Tim Morken" <timcdc@hotmail.com> (by way of histonet)
To:histonet@histosearch.com
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Peter wrote:
<Can backround in IHC be caused by poor fixation or any other part of the
tissue processing?>

Peter,

Lack of proper fixation can affect background staining. The condition of the
tissue before fixation can also affect staining. Necrosis or autolysed
tissue will show spurious staining. One way to check is to run Vimenten as a
fixation control. Poorly fixed or over-fixed tissue will be negative.
Increasing amounts of enzyme pretreatment or heat induced antigen retrieval
to get staining indicate overfixation.

By the way, did you perform a heat antigen retreival technique? That can
lead to generalized background if overdone.




----Original Message Follows----
From: Peter Poon <peterp@cafe.berkeley.edu>
To: histonet <histonet@pathology.swmed.edu>
Subject: Immunostaining
Date: Thu, 13 Jan 2000 15:24:56 -0800 (PST)

Can backround in IHC be caused by poor fixation or any other part of the
tissue processing? I'm using an ABC kit to stain mouse brain tissue. When
I omit primary antibody, my section is completely blank. I've tried three
different rabbit polyclonal antibodies and I've blocked with peroxide,
goat and rabbit serum. In every case the backround is unchanged. When I
decrease primary concentration, the backround and signal decreases.

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