Re: antigen retrieval & Enzyme

From:Mikael Niku

>Some where along the way I was given the impression that enzyme digestion
should always be done before >HIER.  Can you biochemies explain this?

Well, I'm a biochemist by training and I always do HIER first, then enzyme
digestion if necessary. This seems to work better at least in my hands. I
think tissue morphology is better preserved in this order. If you first
erode the tissue with proteases, it can't stand the boiling as well as the
intact fixed tissue.

I'm actually using this combination mainly for genomic in situ hybridization
and not so often for immunohistochemistry. I think (although this is pretty
speculative) that performing HIER first is good for retrieving nuclear
antigens (or accessing the genome, as in this case). The HIER affects the
tissue section in its whole depth, by breaking fixation crosslinks. So, the
tissue will be more porous, and the nuclear targets may be more accessible
for the protease, which otherwise might act most efficiently on the surface
of the section.

   Mikael Niku             URL:
   University of Helsinki  Dept. Basic Veterinary Sciences
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