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@bcm.tmc.edu >
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