Fluorescent In-Situs

[Bruce Appel]

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


Eric,

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

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