B-galactosidase assays

[kovalige at muohio.edu]

I am trying to do B-galactosidase assays on Drosophila embryos and 
have so far been unsuccessful.  The embryos carry a lacZ reporter 
construct that is expressed throughout the latter half of 
embryogenesis and which gives a fairly specific staining pattern.  I 
dechorionate the embryos in bleach, then fix them 20 minutes in 5 ml 
heptane and 5 ml fix mix (100 mM PIPES, 2 mM MgSO4, 1 mM EGTA, 4% 
formaldehyde).  I then wash the embryos three times in 1X PBS 
containing 0.3% Triton X-100.  I add staining solution (1X PBS, plus 
10 mM each potassium ferrocyanide and potassium ferricyanide, 0.3% 
Triton X-100, 0.2% X-gal).  I have stained the embryos overnight on a 
shaker and get no staining whatsoever.  All my solutions are made 
fresh, and the X-gal was purchased recently.  Is there some trick to 
this procedure that I'm not aware of?  I've looked at a number of 
protocols for X-gal staining, and they all seem to be fairly 
consistent, with only minor variations from one another.

Does anyone have any ideas?  I would be very appreciative of any 
suggestions.

Gae Kovalick
Department of Zoology
Miami University
Oxford, OH  45056

kovalige at muohio.edu