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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@bcm.tmc.edu
>
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
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