Hello Gayle, Sorry about the confusion. Yes, I'm detecting the
proteins with antibodies in a double staining. I'd like to try
completing staining of the first antibody the way I have been doing
it (unmasking with citrate buffer, quenching, primary antibody,
biotinylated secondary antibody, HRP-conjugated streptavidin, FITC-
conjugated tyramide), then somehow strip that primary antibody before
continuing a similar process with the second primary antibody and
Cy3. Perkin Elmer says the FITC-tyramide will be covalently bound
and should stay on through subsequent steps.
Do you know a good way to strip the first antibody?
Also, could you tell me more about fluorophores interfering with each
other (part of your message got cut off)?
Thanks, Alice
On Jun 8, 2007, at 11:13 AM, Gayle Callis wrote:
>
> 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
>
>
>
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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----------------------------------------------------------
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