[Histonet] (no subject)

From: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- 
localizeóthat is, they bind each other.  So far, my results make me  
think thereís some physical interference going on between the  
antibodies (and/or the attached biotin-streptavidin, etc)óI see  
mostly Cy3 or FITC, rarely a yellow merge.

Iíve been using Perkin-Elmerís 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 donít 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 itís pretty expensive, so Iíd  
like to hear if anyone has tried this.

Oh, Iíd 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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