Hi, I work in a research lab and have been trying to work out a
protocol for doing a double label for two proteins that really co-
localize—that is, they bind each other. So far, my results make me
think there’s some physical interference going on between the
antibodies (and/or the attached biotin-streptavidin, etc)—I see
mostly Cy3 or FITC, rarely a yellow merge.
I’ve been using Perkin-Elmer’s tyramide amplification for the final
step and really like it for each of these proteins individually (and
in some other double label experiments where the target proteins are
in separate cells or compartments). The company says I should be
able to strip the first antibody-biotin-streptavidin off, leaving
only the tyramide-fluorochrome bound to the tissue, then proceed to
the second antibody. But they don’t have protocols for doing that.
Anyone have some ideas?
Alternatively, Dako suggested I use their polymer system (since the
polymer is very small), with their stripper in between antibodies.
They are not sure if this will work and it’s pretty expensive, so I’d
like to hear if anyone has tried this.
Oh, I’d like to stick to fluorescent signal, and I really have to go
with paraffin-embedded tissue.
Thank a lot,
Alice
Alice Fleming, PhD
Department of Human Genetics
Gonda Building 5524
University of California Los Angeles
Los Angeles, CA 90095
310-267-2456
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