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DAPI staining

Nick nick_owen at wo*ls.demon.co.uk
Sat Jan 31 21:38:34 EST 1998


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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