I wonder if the H2O2 is "poking holes" in the cell membrane, thus allowing
your primary antibody access to the antigen site within the cytoplasm.  I'm
curious if you get the same results with an enzyme digestion pre-treatment
step.

Do you know for sure where in the cell your antigen should be expressed
(cytoplasmic, membrane-bound, nuclear)?

I agree with Laszlo Komuves suggestion of switching to an Alk-Phos system
and testing the H2O2 pre-treatment step.

Good Luck!

*********************************
Catherine "Katie" Bresee Bennett
Sr. Research Technologist
Lovelace Respiratory Research Institute
Albuquerque, New Mexico



> -----Original Message-----
> From:	Elizaha@aol.com [SMTP:Elizaha@aol.com]
> Sent:	Thursday, February 08, 2001 10:05 PM
> To:	Histonet@pathology.swmed.edu
> Subject:	Immunohistochemistry Puzzler
> 
> Hi, 
>     There is an odd problem with an antibody we are using in our lab. It
> is 
> a rabbit polyclonal, we use a streptavidin/biotin (commercial) kit, and 
> chromagen is DAB. Tissue: chondrocytes/cell culture fixed in
> paraformaldehyde 
> 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 reaction 
> like this. Is this weird or what? 
> 
> Thanks again, 
> Elizabeth