FISH for viral DNA

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FISH for viral DNA

Postby ctopherfis » Sep 09 2012 9:14 pm

We've been using biotinylated probes and TSA to detect nuclear viral DNA and can get the proper signal but are having problems with endogenous biotin. We can block with avidin/biotin, but during the hybridization step we loose the block--apparently the avidin disassociates from the endogenous biotin (which is perinuclear/cytoplastic). We apparently loose the block during the DNA denaturation heating phase at 90 degrees C, so we end up with significant background. Fixing with paraformaldehyde after the block and before heat denaturation seems to help but not totally, and it seems to detract from the subsequent hybridization of the probe. We've spent a lot of time on this trying various parameters to no avail. Please if anyone has suggestions on effectively blocking endogenous biotin, and preserving the block during the hybridization phase, I'd like to hear it. Thanks!
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