Re: Immunostaining (background)

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From:<> (Karen Larison) (by way of histonet)
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It should also be noted that polyclonals, unless they have been
affinity-purified to
contain only the antibodies specific for your antigen, almost always give
you more
background than monoclonals.  Also, there is so much batch-to-batch
variability in
polyclonals (unless affinity-purified).  It will depend on what the rabbit
has been
eating, what infections the rabbit has, etc.

Your best bet is to buy a monoclonal that has been shown to cross-react
with the
antigen of interest in your mouse brain.  If you are looking at frog, fish
or other
so-called "lower vertebrates", you are often stuck with using polyclonals.
But if
you are looking at mouse, there's a good chance that a monoclonal is
available that
will cross-react in mouse, particularly if you are looking at brain.  Since
you would
be using a mouse monoclonal on mouse tissue, you may want to buy the Fab or
fragments, although the brain shouldn't have Fc receptors.  Also there are
commercially available blockers that are specifically designed for using mouse
monoclonals on mouse tissue.

Karen in Oregon

Date:          Fri, 14 Jan 2000 09:45:11 EST
From:          "Tim Morken" <>
Subject:       Re: Immunostaining (background)

Peter wrote:
<Can backround in IHC be caused by poor fixation or any other part of the
tissue processing?>


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 <>
To: histonet <>
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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