X-gal Staining Protocol

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(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)
     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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