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Quoting Suzana Glavas <[log in to unmask]>:

> When we visualize the FITC stain, we see nice
> membrane localized staining with very minimal background.  We then switch
> over to DAPI to look at the nuclear staining.  When we switch back to FITC,
> all of the nuclei can now be seen in the FITC channel.  We are visualizing
> with an epifluorescence system prior to doing confocal.  Any ideas what
> might be happening?

First of all, is this happening in the epifluorescence viewing or
only once you get the sample on the confocal?  If it's happening in
wide-field fluorescence I guess it is some photobleaching or activation
effect - try absolutely minimising the UV excitation for the DAPI with
neutral density filters and see if that helps.

If it only happens in the confocal I'd be inclined to suspect
something wrong in the filter changer or spectrometer.

                                                     Guy


--
Associate Professor Guy Cox
Electron Microscope Unit, F09
University of Sydney NSW 2006
+61 2 9351 3176