Hello,

I'm conducting a beta galactosidase staining assay for my fibroblasts using X-Gal. I dissolve X-Gal (stock 50mg/ml prepared in DMSO) in the staining buffer at a final concentration of 1mg/ml. However, even after 24-48 hours of incubation at 37°C (w/o CO2), I'm unable to observe any staining. Additionally, X-Gal precipitates in the dish with prolonged incubation, forming crystals as shown in the picture.

I have also tried heating the staining solution to 65°C before adding X-Gal, but nothing seems to work. Kindly help me resolve this issue.

Composition of my staining solution: 5mM potassium ferrocyanide, 5mM potassium ferricyanide, and 2mM MgCl2 in 1x PBS (pH 6).

For fixation, I use 4% PFA in 1x PBS for 5-10 minutes followed by PBS washes twice before adding the staining solution with x-gal.