I have fixed cells in 3.7% formaldehyde for 30 mins at 30degC, (90 ul
cells, 10ul 37% formaldehyde), spin down and remove supernatant.
Resuspend in PBS+1% TritonX100, spin and remove sup again. Resuspend
in PBS+1% Sodium azide to a final volume of 10ul. Place on
poly-l-lysine slide about 3 ul and allow to air dry. Place a small
drop of Vector Shield (containing DAPI) on the dried cells. Cover slip
and view.
Hope this helps
Nick
On 27 Jan 1998 12:29:57 -0800, mgoodin at nature.berkeley.edu (Michael
Goodin) 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>>
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