Re: protein concentration needed for IHC

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From:<> (Karen Larison)
Date:Thu, 30 Sep 1999 10:22:47 +0000


If things were so simple!  Often things that stain in Westerns or ELISAS 
won't stain in tissue and vice versa.  In tissue, your epitope may be buried 
in the fatty interior of the protein, which has been crosslinked extensively 
with aldehyde fixatives, making the epitope inaccessible.  Or it may be that 
your antigen is a diffusable protein that diffuses out of the tissue before 
it's adequately fixed.  Another possibility is that the epitope may have an 
amino acid residue that reacts with your fixative, thereby destroying its 
antigenicity.  It could be your epitope is a conformational epitope, and any 
manipulation you do that perturbs the conformation will destroy the 
antigenicity, ie running an SDS gel.

Are you fixing your tissue?  You might try fresh frozen sections, or you 
might try some other fixatives.  I have one antibody that works wonderfully 
on Carnoy's fixed tissued, and not at all on aldehyde-fixed tissue.  If you 
are really interested in making the antibody work in tissue, you're going to 
have to try lots of things and hope something works.

Karen in Oregon

Date:          Thu, 30 Sep 1999 08:30:51 -0400
From:          Marcia Bentz <>
Subject:       protein concentration needed for IHC

Hi All,
I was wondering if anyone has ever experimented with what protein
concentrations are detectable in IHC. My problem: an ELISA performed on
lysed supernatent from the same tissue I'm staining showed "off the scale"
for TNF alpha levels, measured in pg/ml. My TNF IHC shows just about nill
in terms of staining. I'm pretty much assuming it varies for each antibody,
but does anyone know a general baseline?
Thanks for the great help!
As always....


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