X-gal Staining Protocol
List of reagents is not yet complete
(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)
- 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
- 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
Relevant papers and books
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Error fetching PMID 13718526:
- Error fetching PMID 6947258:
- Error fetching PMID 13718526:
- Mark Ptashne. A genetic switch. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2004. isbn:0879697164.
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