FISH problem

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Wed May 31 08:44:29 EST 2000


I'm no expert, but I did try a bit of FISH with a FITC labeled probe.
Actually what I did was to use 4 ~50-mers each labeled in 4 places.  From
my reading of the literature, it is my impression most people do use
multiply labeled oligos (although the fluorescent molecules need to be
about 10 bases apart for to prevent quenching, I guess).  I had some
success with this, although my time was limited and I didn't have time to
fully pursue those experiments and work out all the bugs.  I did have some
problems with the rapid photobleaching of FITC.  This was in part helped by
using a commercial stabilizing mounting medium (I used something from
Molecular Probes).

I had the probes directly synthesized with Fluorescine, but if I were doing
the experiments again, I would use a different, more stable label.  In
fact, what I would do would be to synthesize the oligos with a
derivatizable group to which I could latter attach whatever label I wanted.
This would add more flexibility to the experiments down the road.

I hope this helps.

Mike



>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.
>
>
>--
>\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
>   Mikael Niku             URL: www.helsinki.fi/~mniku/
>   University of Helsinki  Dept. Basic Veterinary Sciences
>       - Mit”k– mielt” olen l”nsimaisesta sivistyksest”?
>         Minusta se olisi erinomainen ajatus!
>                                              - Gandhi
>////////////////////////////////////////////////////////////


Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone


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