[Histonet] (no subject)

[Alice Fleming]

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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IMPORTANT WARNING:  This email (and any attachments) is only intended for the use of the person or entity to which it is addressed, and may contain information that is privileged and confidential.  You, the recipient, are obligated to maintain it in a safe, secure and confidential manner.  Unauthorized redisclosure or failure to maintain confidentiality may subject you to federal and state penalties. If you are not the recipient, please immediately notify us by return email, and delete this message from your computer.
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