Chromogenic detection of beta-gal activity can be performed on cells cultured in plastic tissue culture dishes or on glass coverslips. Cells are fixed for 4 min in cold (4°C) 4% paraformaldehyde in PBS and rinsed for 5 min, 2 times. A stock solution of X-gal (5-bromo-4-chloro-3-indolyl b-D-galactopyranoside, Sigma, 40 mg/ml in dimethyformamide, stored at -20oC, protected from light) is diluted to a final concentration of 1 mg/ml in 5mM K3Fe(CN)6, 5mM K4Fe(CN)6, 2mM MgCl2 in PBS, applied to cells, incubated at 37°C overnight (shorter times are sufficient for high levels of beta-gal activity) and examined microscopically for blue cells.
Muscle cells to be labeled for fluorescent histochemical detection of beta-gal activity are cultured on sterile collagen-coated glass coverslips (Becton Dickinson), fixed in 4% paraformaldehyde in PBS and rinsed twice with PBS (where they can be stored at 4°C until staining is performed). If cells are to be labeled with antibody as well as Fluor X-gal, cells are blocked for 30 min in PBS + 10% equine serum, incubated for 2 hrs in primary antibody, rinsed for 10 min 4 times in blocking buffer, incubated 1 hr in biotinylated secondary antibody, washed again 4 times in blocking buffer, incubated 1 hr in Cy5-labeled streptavidin (Amersham, diluted 1:100), then washed 2 times in blocking buffer and 2 times in PBS. All immunolabeling steps are performed at 4oC. beta-gal substrate is prepared by diluting a stock solution of Fast Red Violet LB (Sigma, stock is 50 mg/ml in dimethylformamide, stored at -20oC, not totally dissolved at this concentration) to a final concentration of 100 mg/ml and a stock solution of 5-6 X-gal (5-bromo-6-chloro-3-indolyl b-D-galactopyranoside, Fluka, stock at 50 mg/ml in dimethylformamide, stored at -20oC, will change from pale blue to yellow after exposure to light, but this does not appear to affect activity) mixed to a final concentration of up to 25 mg/ml in PBS (decrease concentration if beta-gal activity is strong). A 0.45 micron syringe filter is used to remove any precipitate. The mixture of Fast Red Violet LB and 5-6 X-gal is added to fixed cells and incubated 60 to 90 min at 37oC, then rinsed in 25 ml PBS for 30 min at room temperature. Nuclei may be stained by diluting 4Õ,6-diamidino-2-phenylindole dihydrocloride hydrate (DAPI, Sigma) in PBS to a final concentration of 100 ng/ml, incubating for 10 min at room temperature and rinsing twice for 5 min. Coverslips are mounted in PBS (no glycerol-based anti-fade solution) and sealed with nail polish. Fluor X-gal staining can be viewed with either fluorescein (FITC) or rhodamine (TRITC) filter sets of an epifluorescence microscope (signal to noise peak is 560 nm for weak signals; our rhodamine filter set has a 560 nm bandpass emission filter). The FITC channel gives a better signal to background ratio for weak signals, but strong signals appear to be quenched. Therefore, Fluor X-gal stain is best viewed with TRITC filters.