In message <[log in to unmask]> Confocal Microscopy List
writes:
>         I'm staining spinal cord and medulla's neurons from rats' fetuses,
> 10 days old, with the following primaries antibodies: Rabbit anti Glycine
> 1:2500, 1:4000; Rabbit anti GABA 1:100, 1:200; Rabbit anti GAD 1:300,
> 1:500; and anti-synaptophysin 1:150, 1:300. I'm using FITC 1:100 or
> Rhodamine 1:100 as my secondary antibodies. I keep getting crossover
> between FITC and Rhodamine despite the different dilutions of the primary
> antibodies I have used. Does anyone has any suggestion?
> Thanks in advance
 
What rhodamine are you using, tetramethylrhodamine or lissamine rhodamine?  The
former will often bleed through, whereas the latter is less likely to do so.
However, it also depends upon the filters that are being used, since even the
most ideal fluorophore may still bleed through a filter if the bandpass of the
filter allows it to do so.  Check the emission spectrum of the fluorophore that
you're using and compare it to the transmission spectrum of the filter--that
should help you determine the source of the problem.
 
Good Luck!
 
Martin Wessendorf
Director, Confocal Microscopic Facility
Dept Cell Biology and Neuroanatomy
University of Minnesota