Our experience with crossover when using FITC with other fluorochromes is
echoed precisely by Marty's advice. Lissamine rhodamine works well with
FITC, but CY is less satisfactory because of the bleed through.
 
We have recently been using the ELF substrate (Enzyme-Labeled-Substrate)
method with the ELF-alkaline phosphatase reagents from Molecular Probes
for double fluorescence immunolabeling. ELF is a phosphatase substrate
that is fluorescent rather than chromogenic. It produces a green
fluorescence with low background. We have found that when it is used with
CY3 in double labeling protocols, there is no bleed through of either
fluorochrome. The ELF step must be done last, however (so far as we can
determine), since the ELF-AP reaction produce seems to be water soluble.
 
Our application is dual fluroescence labeling using a Hamamatsu C4880
fast cooled CCD camera with an MCID image analysis system. I don't know
if ELF would be suitable for Confocal applications. Has anyone tried it
for this purpose?
 
Denis G. Baskin, Ph.D.
Director, Morphological Analysis Core Laboratory
VA Medical Center, Seattle
_____________________________
 
On Mon, 20 Mar 1995, Martin W. Wessendorf wrote:
>
> As near as I can tell, you're absolutely right..._if_ you're talking only about
> FITC showing up through the rhodamine channel.  However, the red fluorophore can
> also be imaged through the green channel.  This seldom is a problem using Texas
> Red or Lissamine rhodamine (--the FITC filter is usually sufficiently
> restrictive).  However, some other red fluorophores (e.g. tetramethylrhodamine
> or cyanine 3.18) have emission spectra close enough to that of FITC that they
> can be seen through the green emission filter.  Thus single-labeling with one of
> those could look as if it were double-labeling, showing up using both the red
> and green filters.
>