Re: IHC Double staining

From:Barbara Wright <bwright@gene.COM>


I have been successful at double labeling with two monoclonal antibodies by doing the sequentially.  Do your first procedure all the way through the substrate (DAB); Start the next procedure by blocking the endogenous peroxidase to rid of all the remaining peroxidase from the HRP step in the first procedure then carry on with your second procedure.  If both antibodies require the same antigen retrieval it is only necessary to do it on the first protocol.  If it requires different antigen retrievals do your HIER as the first procedure and the enzymatic
digestion for your second procedure.  Another tip is if one antibody is localized in the nucleus and the other either a membranous or cytoplasmic antigen, do your nuclear localization first.  I have been successful using two HRP systems and using different colored DAB substrate.  If you need further assistance please don't hesitate to ask. Hope this helps.

Genentech, Inc
South San Francisco, CA

Bruce Abaloz wrote:

> Histonetters,
> I am a student in the department of Zoology at the University of Melbourne, Australia.  I have two antibodies raised in the same species however I need to co-localize their  expression in the one tissue section.  I have protocols for immuno double staining of antibodies raised in different species, but I can not find a protocol or a kit that would allow me to achieve this with two antibodies raised in the same species.  I am currently using immunofluorescence and would like to remain with this visualization.  I would be very grateful for any help.
> Thanks
> Doug
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