In article <41vgrt$b7f at lyra.csx.cam.ac.uk>, john sinclair
<js at mole.bio.cam.ac.uk> wrote:
> We have been using Molecular Probes Fluoreporter kit to detect cells
> transiently transfected with Lac Z constructs with some success.
I invented the technique, so perhaps I can be of some assistance. Good to
hear it still is being used.
> Does any one know if (i) you can load the cell with FDG AFTER
> they have been fixed in ethanol rather than loading them unfixed or
> (ii) is it possible to fix the fluorescein in the cells after
> the FDG has been converted to fluorescein by ethanol fixing.
No. You cannot. The method relies on differential retention of
fluorescein in cells at 0 degrees as compared to 37C. If you rupture the
cell membrane all the fluorescein will leak. If you want to do a
fluorescent staining of lacZ in cells either do an antibody staining or
try a fluorescent precipitating dye system (I think MolProbes makes one.
> Hope somebody can help.
> John S.
Hope that helps