X-gal Staining Blues -- Why Ain't My Cells Gettin' Blue?

Michiaki Masuda masuda at fcs280b.ncifcrf.gov
Tue Nov 30 20:23:33 EST 1993

    I'm trying to stain NIH3T3 cells which are transfected with a plasmid
containing the lacZ gene (derived from pCH110) under the control of a
retroviral LTR.
    I seeded the cells in a 60 mm culture dish and tried to stain the cells
by the following method:

[1] Rinse the cells with cold PBS twice (4 ml each time).
[2] Fix the cells with 3 ml of 2 % formaldehyde / 0.2 % glutaraldehyde
    in PBS for 15 min at 4 C.
[3] Rinse the cells with cold PBS twice.
[4] Add 3 ml of PBS containing 5 mM potassium ferricyanide /
    5 mM potassium ferrocyanide / 2 mM MgCl2 / 1 mg/ml X-gal.
[5] Incubate the culture dish at 37 C overnight. 

   Essentially, I didn't see any difference between the transfected NIH3T3
cells and the untransfected control. Although the possibility cannot be
excluded completely that there is something wrong with the plasmid or
the transfection procedure, results of other experiments sugget that the
staining procedure is the prime suspect.

   I'd appreciate it if someone could provide me with his/her protocol
working successfully and/or some secret tips to help me out from...

   X-gal staining blues...
   X-gal staining blues...
   X-gaaaal staaaaaiiiining bluuuuuues...

   Thank you.

Michiaki Masuda
Laboratory of Molecular Oncology
National Cancer Institute
Frederick, MD 21702-1201
E-mail: Masuda at ncifcrf.gov

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