>What filters are you using to get complete separation of FITC and Cy3, and
>what line are you using on the argon laser to excite these fluorochromes?
>Leslie T. Malmgren, Ph.D.
>SUNY Health Science Center
>750 East Adams St.
>Syracuse, N.Y. 13210
>(315)464-7254
 
First of all, you have to fiddle a bit with the filters depending upon what
the material is you are working with, so I will give you a broad outline of
what we have found to work, taking into account how cleared the tissue is,
presence of antifade medium, etc., but all that aside, the Cy3/FITC combo
seems very forgiving.  We have excited FITC-Cy3 with the 488, 514 and all
lines, depending upon the overall intensity of staining of either one of
the fluorochromes (if the FITC is, relative to the Cy3,too bright, then use
the 514, if the FITC signal is weak or faded, use the 488.  I use a 525
barrier filter, then generally a 535/595 beam splitter for the two
detectors.  If required, I may add another couple fiters in front of the
detectors (an FITC narrow-band pass filter and a 600 long-pass filter for
the Cy3 detector.  Given the intensity of the two fluorophores, we can
typically backoff on the PMT's and generally end up with completely
separate stacks.  We are still experimenting with it, but atleast for us,
it was a major improvement in the quality of our double labeling procedure.
 The only time we got the FITC-Rhodamine to look good was when the
rhodamine signal was extreme (neurons intracellularly filled with
rhodamine-dextran) combined with FITC labeled processes (nerve terminals,
receptors, gap junctions, etc.) on these intensely labeled cells.  Although
we have purchased some, we have yet to test Mol. Probes Bodipy (narrow
emission band replacement for FITC) fluorochrome, given that the ole FITC
is working fine, but if you still have a problem, you may want to try the
Bodipy with Cy3.
 
Anyway, give it a try, I think you will be impressed.