FISH problem

Mikael Niku mikael.niku at helsinki.fi
Wed May 31 05:29:48 EST 2000


Hello!

I'm using an in situ hybridization protocol utilizing DIG-labeled
probes and alkaline phosphatase visualization to detect
genomic sequences in tissue sections and blood spins. This is working
excellently.

Now I'd like to do the same thing with FITC-labeled probes.
The probe sequence is the same, the protocol is basically
the same (before visualization steps, of course) -
but I don't get any signal.

I haven't done FISH before but I have used fluorescently
labeled antibodies, so there should be no problems in
the basic handling of fluorescent components. The probe
is commercially made and directly labeled with FITC.
I have tried with two separately prepared lots, with
no success. The in situ hybridization protocol is pretty
standard.

Any ideas?

How about stability of the FITC label in denaturation,
hybridization and washing steps? And how about sensitivity as
compared with enzymatic detection? I get a strong signal with
the AP system. For FISH, I have one FITC label per an oligonucleotide
probe.

It is of course possible to get an anti-FITC antibody
and try to detect the probe that way. I haven't done this
yet (and that's not what I would like to do routinely, as
I'd like to keep the protocol quick and easy). Just decided to
try first if someone here has clever ideas as to what I might be doing
wrong.


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   Mikael Niku             URL: www.helsinki.fi/~mniku/
   University of Helsinki  Dept. Basic Veterinary Sciences
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                                              - Gandhi
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