b-gal staining of fixed/unfixed cells

Martin Leach leach at mbcrr.harvard.edu
Tue Oct 12 10:13:46 EST 1993


Dear all,
had to post this protocol to someone, so as I typed it in I though I would
post it here.

It is taken from C.Fulwiler at the MRC in Camb. England 3/91.

Here Goes:

Wash cells in PBS
Fix cells for 10' in a 1:20 diln of 2.5% Glutaraldehyde (diluted with PBS).

Make a stock of the stain:

5mM K3Fe (CN6) - potassium ferricyanide (165mg in 100ml)
5mM K4Fe (CN6) - potassium ferro-cyanide (210mg in 100ml)
2mM MgCl2        (0.2ml of 1M stock in 100ml)
0.01% Triton X-100 (1ml of 10% stock in 100ml)
Make up to 100ml with PBS
Freeze aliquots at -20C

For staining place 315ul of 40mg/ml X-gal (in DMF) and 315ul of 0.1M IPTG
per 8.5 ml of stain.(Add fresh) per 100mm dish. Prewarm this mix at 37C
then filter thro 0.4micron to remove particles. If this is omitted large
crystals precipitate and grow on the culture dish. Place stain on culture
and leave in 37C incubator for at least an hour.

Should see nice blue cells.

Good Luck

Martin Leach.

p.s. someone mailed me saying that they tried the suggestion of just adding
x-gal and IPTG to a dish of cells, and they stained!! (Without fixing). I
would suggest 120ul of X-gal and IPTG per 10ml of medium

-- 

.....          Martin Leach                Email:leach at mbcrr.harvard.edu 
   _|____      Dept. of Pharmacology       Phone: (617) 638-5323        
   / o  /      Boston Univ. School of Med. Fax:   (617) 638-4329         
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(BULLDOZER) \_ Boston MA 02118            "Not the old underpants on your
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