mouse frozen sections/ihc stianing

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From:Gayle Callis <uvsgc@msu.oscs.montana.edu>
To:histonet@Pathology.swmed.edu
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Date:Mon, 10 May 1999 08:45:21 -0600
Content-Type:

When you are staining frozen sections of mouse tissue, often you cannot
get an antibody that is not a rat antimouse, hamster antimouse or even
mouse antimouse.  This is very apparent in catalogs providing monoclonals
to mouse cell surface markers (CD's, etc).

There are ways to avoid background fluorescence with rat, hamster antimouse.
Many of our monoclonals are biotinylated and when using purified antibodies,
ascites or supernates, dilution panels must be done on

1.  Primary (supernates are usually put on section without dilution) 
2.  Secondary antibody (can be too concentrated!)
3.  Strepavidin-fluoresent labels (Strep avidin-Alexa 546 and 488 will give
background unless diluted out, with our acetone fixed sections up to 1:1000)
This is also true of other labels, the specification sheets give dilution
ranges for IHC or at least my Jackson ImmunoResearch spec sheets do this,
or ask your vendor.

We also like to use F(ab')2 fragments for secondaries, even with a fluorescent
label


It also pays to dilute the secondary in normal blocking serum (5, 10% serum)
and even perform a block before the primary.

Power outage coming on, and not done blabbing

Gayle Callis



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