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confocal microscopy, fluoro beads, 3 types of cells on one slide - (Apr/28/2005 )

Hi folks,
I am struggling with confocal microscopy of tree different cell type grown together on lysine coated chamber slide.
1 Transfected cells with HA-tagged gene of interest (fibroblast like cells), secondary antibody green
2. untransfected cells of the sam type
3. MRC-5 cells marked with fluorescence beads (blue) untransfected for control of second protein of interest.

I use DAPI as counterstaining for nucleus. I also look for level of other protein in all tree cell type (secondary antibody red).

I have a problem
1. Beads have such a strong signal that I cannot acquire DAPI without saturating beads.
2. I have “bleed trough” of beads signal also in red and especially in green channel.
I use confocal microscope from Leica equipped with Leica software.

Is there any possibility to avoid this kind of artifacts and to improve my image?

Comments and advises are appreciated
Beforehand I thank you



Where are your beads from - what company? Are they supposed to be specific for the blue diode laser? If these cells are purely for a control for your second protein of interest (which if I understand correctly you are staining with an antibody in red) why mark them with beads at all? Do you really need all three cell lines on the same well? Can you put them into different chambers on one slide for your controls? This is what I do with great success.