You are probably experiencing autofluorescence induced the aldehyde 
fixation.  You can try Molecular Probes Image - IT image enhancer that is 
supposed to get rid of the autofluorescence.

It is important to note that Prolong Gold antifade is supposed to sit 
overnight and not sealed with fingernail polish, by curing overnight, this 
mounting media, the refractive index gradually increses as it cures.   This 
may be why the control appeared black just after you mounted the coverslip.

You can tell the CLSM to get rid of the autofluorescence too, some might 
consider this cheating, but it can be done.   What may be better is to NOT 
fix the cells, and view them with CLSM - GFP is designed for use with 
living cells and you may find the GFP is even brighter.  The Clontech 
manual called Living Colours tells you a great deal about handling cells, 
etc with GFP.  That manual can be accessed online in pdf form.

Unfixed cells can also be mounted with Prolong Gold antifade w/ DAPI - we 
do this with unfixed frozen sections to avoid all fixation or you can add a 
DAPI solution to the unfixed cells, and view them without coverslipping or 
coverslipping with PBS.

    At 11:13 PM 6/6/2006, you wrote:
>Hey,
>
>I have a GFP labeled g-protein coupled receptor that I am expressing
>in HEK 293 cells.  I cultured the cells on collagen coated glass
>chamber slides, fixed the cells with 4% PFA, and then coverslipped
>with Prolong Gold antifade containing dapi.  Before I coverslipped I
>checked the slide on a standard fluorescent microscope to make sure
>everything was expressing correctly.  My transduced cells had a nice
>signal and my control was black.  The day after coverslipping my
>control cells showed a noticeable green signal, not quite a bright as
>the GFP cells, but it is apparent.  I took confocal pics and there
>was clearly some green in my control cells.
>
>Does anyone have a suggestions?  I figure one of two things
>happened.  Either some cells slid around when I pressed down on the
>coverslip or maybe I am getting some sort of bleedthrough.  I am not
>very good with the confocal yet, so I don't know if I did something
>wrong here.  Does anyone have a good protocol for growing 293 cells
>on glass slides?  I have heard there of some super adherent clones of
>293 that I would like to try, although I haven't found a source for
>them.
>
>Any and all suggestions would be appreciated.
>
>Thanks,
>
>Caroline
>
>
>
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Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)



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