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X-Gal Staining Protocol for beta-Galactosidase
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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:


      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).


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.


      Sites of enzyme activity -------------------- blue

      Nuclei ----------------------------------------- pink

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