Fluoreprter LacZ detection

Garry P. Nolan gnolan at cmgm.stanford.edu
Sun Sep 3 12:21:05 EST 1995


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


Garry Nolan



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