X-gal Staining Protocol

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Contents

Overview

List of reagents is not yet complete

Procedure

(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

References

Relevant papers and books

No further references available at this time

Contact

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