2015-07-06 LM
Description: This method is used for the detection of β-galactosidase on
formalin-fixed or fresh frozen sections, and it is NOT suitable for
formalin-fixed, paraffin embedded tissue sections. The sites of βgalactosidase 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
(aliquots stored in box #72).
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 (if fresh frozen). If formalin-fixed sections are used, dry
the slides overnight at RT.
2.
3.
4.
5.
6.
7.
8.
Wash slides with 3 changes of PBS for 5 minutes each and then rinse
in distilled water.
Incubate slides in X-gal working solution at 37 C for 24 hours (use
humidified chamber to prevent slides from drying).
Rinse sections in PBS for 2x5 minutes
Rinse with distilled water briefly.
Counterstain with nuclear fast red for 3-5 minutes.
Rinse in distilled water.
Mount DIRECTLY with aqueous mounting medium.