Hi folks,
I'm using GFPfusions to study nuclear import in yeast. The GFP
expression is tremendous however getting good DAPI staining is a problem.
I've tried 3.7% formalin, 4% paraformaldehyde in water, 4%
paraformaldehyde in PBS. I've used DAPI at 0.5 and 2.5 ug/ml. Fixation
was done at for 15min to 2h at RT on a rocker shaker. Formaldehyde
fixation gets the DAPI into the cells but severly compromises GFP
fluorescence. Paraformaldehyde fixation is easier on GFP but the cells
don't take up DAPI. Oh one more thing, fixation in 50% EtOH kills GFP.
Any suggestions/protocols would be greatly appreciated.
Cheers,
Michael Goodin
mgoodin at nature.berkeley.edu
