RE: Immunohistochemistry Puzzler

I agree with Geoff, the h202 is an enzyme which can digest some of the ends
of the molecule which may be masking binding of the antibody to the antigen
, such parts of the molecule include chondroitin sulfate,  and keretin
sulfate, I stain chondrocytes all the time and use extensive digestion
methods to get at the antigen site, on the other hand this may be binding to
your secondary antibody as Geoff suggests I would use the h202 and leave out
the primary antibody and see if you get the same staining, but if your
staining is chondrocyte specific I doubt that non specific binding of the
secondary is the problem.
Patsy Ruegg
U of Iowa Hybridoma Bank sells some great antibodys to chondrocytes (LINK
Protein, and Collagen)

-----Original Message-----
From: Geoff McAuliffe
Sent: 2/9/01 7:59 AM
Subject: Re: Immunohistochemistry Puzzler wrote: 

    There is an odd problem with an antibody we are using in our lab. It
a rabbit polyclonal, we use a streptavidin/biotin (commercial) kit, and 
chromagen is DAB. Tissue: chondrocytes/cell culture fixed in
and embedded in paraffin.. 
Problem:  When we stain cells with it we get little to no label, if we
add a 
hydrogen peroxide blocking step before the primary antibody we get heavy

labeling of cells that looks specific/intracellular. The antibody is
made "in 
house" but is not against hydrogen peroxide? Has anyone seen any
like this. Is this weird or what? 

Thanks again, 

Dear Elizabeth: 

    My guess, and I do mean guess, is that the peroxide is "etching" or
"opening up" the antigenic sites to make them more available for
binding. Hydrogen peroxide treatment can dramatically speed up the
staining of EM grids by etching the surface of the epoxy sections. What
happens when you use the peroxide block but omit the primary antibody? 
    Are you getting all of the wax out of the sections? 

Geoff McAuliffe, Ph.D. 
Neuroscience and Cell Biology 
Robert Wood Johnson Medical School 
675 Hoes Lane, Piscataway, NJ 08854 
voice: (732)-235-4583; fax: -4029 

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