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Hi,
I believe that you either have an old Vectashield or you are leaving too 
much water on coverslips before mounting them. You may try Prolong, or 
Prolong Gold they both are much more efficient (than Vectashield) 
anti-bleach mounting media, their disadvantage is that they dry and shrink 
cells a little bit. Therefore for a high resolution 3D microscopy I would 
use Vectashield. But if you are not interested in the 3D rendering Prolong 
(Prolong Gold) is much better.
Regards,
Art

--On Friday, November 17, 2006 12:22 PM +0100 Karl-Johan Leuchowius 
<[log in to unmask]> wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> 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
>
>




Artem Pliss, Ph.D.
Department of Biological Sciences
State University of New York at Buffalo
Cooke Hall #628, Buffalo, NY, 14260
Phone: 716-645-2363 (ext 156/157)
Fax: 716-645-2975