X-gal Staining Protocol
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(Bruce Conklin and David Sanan, Gladstone/UCSF)
* 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
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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