[Histonet] dapi and GFP problem problem

From:Caroline Bass


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  

Any and all suggestions would be appreciated.



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