PI and GFP

David Galbraith dgalbrai at ag.Arizona.EDU
Mon Feb 24 21:32:17 EST 1997


Wendy:

I think ethanol fixation eliminates GFP fluorescence -- it disrupts the
beta-barrel and lets solvent into the fluorochrome, thereby quenching it.
I'd try other fixatives, such as paraformaldehyde, and try to keep GFP in 
a nearly-native environment. Alternatively, maybe
you could get the PI in through electroporation (?)

David Galbraith

On Mon, 24 Feb 1997, Wendy D. Schober wrote:

> 
> We are attempting to optimize the procedure to label transfection along 
> with cell cycle in HeLa cells.  So far we have co-transfected with CD20 
> and then labeled the surface with CD20-FITC.  We found 5-13% CD20 
> positive with good PI patterns after the usual 70% ethanol fixation.  
> We used the same cells and fixation procedure with GFP as the reporter and 
> hoped to find similar results.  Unfortunately, there were few cells GFP 
> positive and what may be there are of very low intensity, barely over 
> background.  We are looking for suggestions for improving our GFP and keeping 
> good PI patterns.  The GFP is from Invitrogen and they have been somewhat 
> helpful, but have not done this with PI.  
> Is fixation causing a problem??  If so, is there a better procedure which 
> will permeabilize and fix to get both GFP and PI to work?? Ultimately, we 
> want this to work in some fibroblast lines, but HeLa is our control.  
> 
> Thanks in advance for any hints.
> Wendy Schober
> wschober at bcm.tmc.edu
> 




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