1. Remove cell media, wash cells twice with PBS.
2. Fix cells in PBS/0.05% Glutaraldehyde. Incubate at room temperature for 5 minutes.
4. Remove PBS/0.05% Glutaraldehyde and wash twice with PBS.
5. Stain with PBS containing
1 mM MgCl2,
5 mM K4[FeII(CN)6],
5 mM K3[FeIII(CN)6],
1 mg/ml X-gal. X-gal staining solution should be warmed to 37 °C for 5-10 minutes before use.
6. Incubate at room temperature or 37 °C in dark until desired level of
staining is observed (several hours to overnight). Stop staining by washing several times with PBS.