In message  <[log in to unmask]> Roland Rosqvist writes:
> I am having problems with scanning Cy5. It fades extremely quickly. This goes
> against common experience, I guess. I am labelling bacteria with an antibody
> against a membrane protein followed by a secondary ab labelled with Cy5. The
> antifades I've used are Citifluor and PPD, with the same bad result in both
> cases. There are cells present which have been fixed with PFA and are in a
> glycerol-antifade mount medium.
>
> Since I'm scanning two signals (FITC and Lissamine Rhodamine) first and
> subsequently the Cy5 signal (using a KrAr laser), the Cy5 is being faded by
> the
> 488/568 excitation. This seems to go against theory, since these lines
> shouldn't affect Cy5 in any way (?). Please comment.
 
If you can mount in DPX (Fluka) rather than in PPD, that may work better--my
sense is that it fades less that way (although I've never performed the
experiment).  However, DPX is not good for FITC.  Bodipy is green and works well
under DPX.  However, bright Bodipy is visible under rhodamine filters and thus
isn't great if you have single cells that are double-labeled.
 
The 568 nm line will excite Cy5 at about 15% of it's maximum--this is why you
need to use a barrier filter that blocks it out, if you're using both rhodamine
and Cy5.  Depending upon the neutral density filters that you're using, you may
be damaging Cy5 more with the 568 nm line (or even the 488 nm line, which
excites Cy5 at about 1% of its maximum) than by exciting it with its intended
line (647 nm).
 
Good luck!
 
Martin Wessendorf