fix and permeabilize Cells
(by ucgatan from ucl.ac.uk)
Tue Jul 10 07:36:51 EST 2007
On Mon, 9 Jul 2007, aakash wrote:
> I would be glad if any of you can share your experience about how to fix
> and permeabilize GFP expressing cells without quenching/killing the GFP
> for cell cycle analysis using PI. GFP is not fusion protein. I used
> paraformaldehyde and ethonol, but i loose complete gfp expression.
You don't say what analysis method this is for; i'm guessing flow
I don't do that myself, but i do do fluorescence microscopy with GFP. We
fix in 4% methanol-free formaldehyde in a fancy buffer; it should be
around pH 6.8, but a quick measurement puts my tube at about pH 5.0. It
still seems to work okay! PBS at pH 7.4 is basically also fine, so i guess
pH is not absolutely crucial at this step. We fix for 10-30 min. We
permeabilise with 0.5% Triton X-100 in PBS, for 10 min.
Anyway, GFP fluorescence seems fine in those conditions.
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT
(t) +44 (20) 76797264 (f) +44 (20) 76797805 (e) thomas.anderson from ucl.ac.uk
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