Hmm, I've seen good GFP fluorescence in my fixed cells (3.7% formaldehyde
in synthetic medium, also in YPG, fixed about 1 hour on the shaker at
30C). My fluorescence diminishes with long fixation times, and similarly
to your results, in methanol. For DAPI, I've only stained live cells, and
then it has been variable. Maybe DAPI stain first and then fix?
Mike Conboy
Cyert Lab
Dept. Biological Sciences
Stanford
In article <mgoodin-2701981113390001 at 128.32.128.241>,
Michael Goodin <mgoodin at nature.berkeley.edu> wrote:
>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>>>
