[Histonet] X-gal staining and fixation

[Tamama, Kenichi]

Dear histonet members,
 
I have been working on X-gal staining of cultured cells with iron cyanide
solution to see the efficiency of adenovirus transfection, and  decaying of
cell structure is bothering me.  I fixed cells with 1%glutaraldehyde/PBS(-)
for 30 min followed with 0.2% Triton-X/PBS(-) for 10 min before incubation
with iron cyanide solution.  The cell structure looks fine soon after
fixation, but some junks/debris of cells start floating soon after
incubation with X-gal solution.   Higher conc of glutaraldehyde preserves
the structure more robustly, but at the same time it denatures the
beta-galactosidase and loses the blue color. 
 
I would deeply appreciate your comments/suggestions/advice.
 
Ken Tamama
 
Kenichi Tamama, M.D.,Ph.D.
Resident of Clinicial Pathology (Laboratory Medicine)
University of Pittsburgh School of Medicine
Department of Pathology