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Karl-Johan,

I've been working with a very similar setup and right now, on a modified
LSM510 (where we moved from inverted to upright position), I fill up the
wells completely with glycerol+10% 10x PBS and I have no more problem like
the ones you talked about (I got exactly the same problem using small amount
of various mounting media - I almost tried them all!!). 
You can also get a better recipe adding anti-fading agent. Let me know if
you want more details about it.

/Laurent

______________________________________________
Laurent Barbe
Royal Institute of Technology (KTH)
Dept of Biotechnology - School of Nano-biotechnology
Albanova University Center, Roslagstullsbacken 21
10691 Stockholm - SWEDEN
tel:+46 855378832
fax:+46 855378323
http://proteinatlas.org/
http://www.biotech.kth.se/nano_biotechnology/

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Karl-Johan Leuchowius
Sent: fredag 17 november 2006 12:23
To: [log in to unmask]
Subject: Problems with confocal microscopy in 96 well plates

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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