On Wed, 22 Dec 1999, Grace Jones wrote: > What is the best way to fix cells expressing a GFP marker, so that one can > counter stain with PI or do some fluorescent immunocytochemistry, but yet > the fix does not kill the GFP marker? Hi Grace, If you just want to use PI as a nuclear marker (rather than quantatively), any aldehyde fixative will do (Paraformaldehyde of formaldehyde are usual). If you are using a fluorescenec microscope it is best to keep the PI concentration low as it is very bright and can overwhelm some low GFP signals. 7-AAD would be an alternative here as the signal from blue excitation isnt as strong. A further alternative is to use a GFP than localises in the cell membrane (eg Clontech's EGFP-F) and then you could use ethanol fixation (normally ethanol fixing will abolish the GFP fluorescence). Good luck, Derek ************************************************************************ Derek Davies Voice: (44) 0171 269 3394 FACS Laboratory, FAX: (44) 0171 269 3100 Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk London, UK Web Page: http://www.icnet.uk/axp/facs/davies/index.html In tenebris lux *************************************************************************
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