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Fluorescent In-Situs

Bruce Appel appel at uoneuro.uoregon.edu
Fri Jun 2 15:29:00 EST 1995

In article <3qlpv4$67r at vixen.cso.uiuc.edu>, Eric Spana <e-spana at uiuc.edu> wrote:

> Hello out there-I've been looking for a protocol to do in situ 
> hybridizations to fly embryos using a fluorescent signal and confocal 
> detection.  I've always been taught that the signals from an AP 
> secondary are stronger than HRP which in turn is stronger than 
> fluorescence.  I think this fact/rumor refered to typical fluorescence 
> though, not confocal laser detection and nobody every mentions what 
> fluore (FITC, RITC or Cy5).  I do presume that this is correct since 
> everyone always does their in situs with AP even though the HRP product 
> is more "photogenic".  So the question is-will fluorescent in situs 
> work?
> Thanks in advance,
> Eric Spana
> e-spana at uiuc.edu


I think it's unlikely you'd be able to detect a riboprobe through
more-or-less conventional (say, dig-labeled probe detected by anti-dig
followed by a fluorophore-conjugated antibody, or some variation)
fluorescent detection techniques. However, Molecular Probes (Eugene,
503-465-8300) has developed a fluorogenic substrate for alkaline
phosphatase (kits for both antibody labeling and in situ RNA
hybridization). Peak emission of the product is between 500-550 nm. It's
incredibly stable and quite bright. The downside is that I find it to be
somewhat less sensitive than alk. phos. development by NBT/BCIP and the
product forms a crystalline precipitate, which makes for kind of a
stringy-looking signal. So I think it would be useful in using a probe
that shows a rather broad distribution, but single-cell stuff might be
dicey. I haven't messed with it much, maybe you can do better. In fact,
it's been a year since I tried it; you should call Mol. Prob. and ask,
they may have developed something better.

I'm not connected to Molecular Probes and it's rather unlikely I'll
benefit from the plug.


Bruce Appel
University of Oregon
appel at uoneuro.uoregon.edu

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