B - Galactosidase Staining
1. Wash Cells or tissue sample in room temperature PBS.
2. Prepare the following solutions:
A. PBS - Glutaraldehyde (1.25% solution)
Glutaraldehyde 25% stock (in -20°C freezer) 2 ml
PBS 18 mlNote: Handle stock glutaraldehyde solution with extreme care in the hood!
B. Staining solution
Ingredients For 10 ml
Tris HCl pH 7.5 (50 mM) 500 mL of 1 M stock
Ferriferrocyanide sol'n (2.5 mM) 500 mL of 50 mM stock
NaCl (15mM) 30 mL of 5M stock
MgCl2 (1mM) 10 mL of 1M stock
X-gal (0.5 mg/ml) 250 mL of 20 mg/mL stock
ddH2O 8.71 mLNote: X-gal stock is made in 100% N,N-Dimethyl Formamide and is stored in the -20°C freezer in foil. Stocks may be kept for up to 2 weeks then discarded.
Note: Add ferriferrocyanate and X-gal last, just prior to use.
C. Ferriferrocyanate solution
Potassium Ferrocyanate 210 mg
potassium Ferricyanate 164 mgAdd 5 mL millipore water, keep in foiled tube. Make fresh with each use.
3. Fix the cells or tissue in the glutaraldehyde solution. For cells, fix for 5 minutes.
For tissue, mince such that particles are at most 1 cm3 in size, then fix for 10 minutes.
4. Rinse cells or tissue twice with PBS without Calcium/Magnesium (Calcium inactivates the X-gal conversion process).
5. Remove all of the PBS and add the staining solution. Incubate at 37°C in the incubator. Incubation should be in the dark as the X-gal substrate is light- sensitive. For cells, can see blue in 4-6 hours. With tissues, incubate O/N.
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