Protocol
Materials
Solutions
PBS
0.05% glutaraldehyde: Dilute 25% stock 500x in PBS. Stock should be stored frozen at -20C in aliquots
X-gal solution: Prepare in PBS 30 mM potassium ferricyanide (K3Fe(CN)6), 30 mM potassium ferrocyanide (K4Fe(CN)6-3H2O), 1 mM MgCl2 or MgSO4. Add Xgal to 1 mg/ml final concentration just before use. The solution minus the Xgal can be stored a few months at room temp.
Methods
Procedure
1. Discard medium and wash twice with PBS. Add 2 ml fix (0.05% glutaraldehyde) per 60 mm dish and incubate for at least 5 minutes and no longer than 15 minutes.
2. Rinse cells three times in PBS as follows: Wash 1 and 3 are quick washes. Wash 2 is 10 minutes.
3. Add X-gal solution. Use a minimal volume to cover the cells, about 0.5 ml for a 60 mm dish.
4. Incubate from 1 hr to overnight at 37°C. Positive cells will stain blue.
5. Remove stain and overlay with 80% glycerol. The dishes can be stored indefinitely at 4°C
