X-gal Staining Protocol

(Bruce Conklin and David Sanan, Gladstone/UCSF)

Materials:

• Potassium Ferrocyanide Crystalline (Fisher Scientific #P232-500
• Potassium Ferrocyanide Trihydrate (Fisher Scientific #P236-500)
• Magnesium Chloride (Fisher Scientific #M33-500)
• 1X PBS
• X-gal (Boehringer Mannheim #745-740)
• DMSO
• 2% Formaldehyde 0.2% Gluteraldehyde in 1 X PBS
• Superfrost/ Plus Microscope Slides (Fisher #12-550-15)
• Pap pen (Electron Microscopy Sciences #22303)
• Nuclear Fast Red ( also called Kernechtrot)
• Gel Mount (Biomeda, M01)

Solutions:

1) Solution A:
5mM Potassium Ferricyanide Crystalline
5mM Potassium Ferricyanide Trihydrate
2mM Magnesium Chloride
in 1 X PBS
(stored at 4 deg, protected from light, then warm to 37 degrees C prior to using)

2) X-gal Stock Solution (40X):
40 mg/ml in DMSO (100 mg in 2.5 ml DMSO)
(store at -20 deg, protected from light)

3) Final X-gal Solution:
Dilute X-gal stock solution 1:40 in Solution A.
(first warm Solution A to 37 degrees C to prevent precipitation of X-gal)

4) Formalin/Glutaraldehyde fixative:
12.3 ml distilled water
10.0 ml 1% glutaraldehyde
2.7 ml formaldehyde stock (37%)
25.0 ml x2 saline buffer

Staining Protocol:

1. Cut 10 micrometers cryostat sections onto pap-penned slides from fresh-frozen tissues. Immerse immediately into cold formaldehyde/gluteraldehyde 5 minutes, then rinse in dH20 for 60 seconds.
2. Let section dry completely onto slide
3. Rinse with 1X PBS
4. Apply Final X-gal Solution to sections and incubate at 37 deg; for 30 minutes to 24 hours (Check sections under microscope every 2 hours for development of blue staining)
5. Rinse with 1X PBS
6. Wash 2 X 2'' with deionized H2O.
7. Counterstain 3'' with Nuclear Fast Red
8. Wash as in step 6, then cover slip with Gel Mount

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