X-gal Staining Protocol

Overview
List of reagents is not yet complete

Procedure
'''(Bruce Conklin and David Sanan, Gladstone/UCSF)

Materials:'''

Solutions:
 * 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)

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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