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Dear all,

I am trying to do multicolour fluorescent staining (Hoechst 33342, Alexa 
488, Alexa 555, Texas Red and cy5) of cells grown in 96 well plates 
(Nunc CytoWell with cover glass bottom) which I look at with a confocal 
microscope (Zeiss 510 meta, 63x or 40x oil objective). I add Vectashield 
to mount the cells and to protect the fluorophores from fading. This 
procedure works nicely when I use cells grown on slides, mounted under a 
cover glass.

The problem I'm experiencing with the staining in the 96 well plates is 
that the vectashield seems to make everything look hazy--the staining 
looks weaker and the background seems to increase. The more vectashield 
I add, the worse it looks. Also, the haze effect grows stronger over 
time, so that after a few hours all I can see is something that looks 
like "fluorescent fog". I have also tried to do without the vectashield, 
and the cells look great (bright and crisp staining, no background) when 
looking through the lens. However, if I try to look at them confocally 
they look terrible (probably because of optical effects).

Does anyone have any ideas of what to try? Would another mounting 
medium, such as Prolong, work better? How much mounting medium should be 
used?

Thanks for your help!

cheers,
Karl-Johan

-- 
Karl-Johan Leuchowius, M.Sc., PhD student

Molecular Medicine, Dept. of Genetics and Pathology
Uppsala University
Rudbeck Laboratory
Dag Hammarskjölds Väg 20
S-751 85 Uppsala
Sweden

Phone: +46 (0)18 471 4851