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X-Gal Staining Protocol for beta-Galactosidase
Description: This method is used for the detection of β-galactosidase on formalin-fixed, frozen sections, and it is NOT suitable for formalin-fixed, paraffin embedded tissue sections. The sites of β-galactosidase activity will be stained blue and nuclei will be stained pink.
Solutions and Reagents:
Chemicals:
Potassium Ferricyanide Crystalline (Sigma #P-8131, FW 329.2)
Potassium Ferricyanide Trihydrate (Sigma #P-3289, FW 422.4)
Magnesium Chloride (Sigma #M-8266, FW95.21)
X-gal Dilution Buffer:
Potassium Ferricyanide Crystalline (5mM) --------------160 mg
Potassium Ferricyanide Trihydrate (5mM) ------------- 210 mg
Magnesium Chloride (2mM) ------------------------------ 20 mg
PBS
----------------------------------------------------------100 ml
Mix well and stored
at 4 ºC, protected from light.
Warm to 37 C prior to use.
X-gal Stock Solution (4% in DMF):
X-gal (Boehringer Mannheim #745-740) ---------------- 20 mg
DMF (N, N Dimethylformamide) ------------------------- 0.5 ml
Mix until completely dissolved. Store at –20
ºC,
protected from light.
X-gal Working Solution:
First warm X-gal dilution buffer to 37
ºC
to prevent precipitation of X-gal.
Then dilute X-gal stock solution 1:40 in warmed X-gal
dilution buffer (keep buffer warm at 37 C before applying to
slides).
Procedure:
1.
Cut fresh frozen
sections and fix with cold formalin (4 C) for 10 minutes.
2.
Wash slides with 3
changes of PBS for 5 minutes each and then rinse in distilled water.
3.
Incubate slides in
X-gal working solution at 37 C for 24 hours (use humidified chamber
to prevent slides from drying).
4.
Rinse sections in PBS for 2x5 minutes
5.
Rinse with distilled water briefly.
6.
Counterstain with nuclear fast red for 3-5 minutes.
7.
Rinse in distilled
water.
8.
Mount DIRECTLY with
aqueous mounting medium.
Results:
Sites of enzyme activity -------------------- blue
Nuclei ----------------------------------------- pink