[Histonet] (no subject)

[Gayle Callis]

You lost me on this message, can you tell us exactly how you are doing 
this?  Are you detecting the proteins with antibodies, in a double staining?

You may have a problem with quenching (this is NOT photobleaching) where 
the fluorophores interfer with each other in such cloAt 09:20 AM 6/8/2007, 
you wrote:
>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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Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610