> Hello........I am looking for a technique for the simultaneous hyb'ing of
> 2 different bacterial species (occ. found in a biofilm), with 2 different
> fluorescently labeled oligo probes.......preferably using Texas Red and
> Fluroescein as the labels. I have gotten the Texas Red to work alone in a
> pH 6.0 hybridization environment, but am stumped as to how to get the
> Fluroescein probe to work since it needs a pH 9.0 environment.
>
Chris,
I have no direct experience with FISH, but I would suggest you look dump the
Fluorescein (which is notoriously pH sensitive - hence the slight
modification of these dyes to make pH probes !!!!!), and move to
either BODIPY-FL (not related to fluorescein) or better still Alexa
(not Greek, but is insensitive to pH bewteen 4 and 10). These dyes are
available
from Molecular Probes, and are insensitive to pH, more photostable and in
addition
have narrower excitation/emission spectra but with maxima similar to
fluorescein. What more could you want ?
Good luck,
Ian.
PS.
[ I have no commercial interest in Molecular Probes - just a very
satisfied customer].
Their website, which is also searchable, is at http://www.probes.com
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Dr Ian S. Harper
Confocal Microscopy Facility
Department of Biological Sciences
Monash University, Clayton, Vic 3168, Australia
Email: Ian.Harper at sci.monash.edu.au
Tel: +61 3 9905 5635; Fax: +61 3 9905 5613
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