β-Galactosidase staining of frozen sections

The Jackson Laboratory Cre Repository uses β-galactosidase staining of fresh-frozen sections for 15.5 dpc embryos, postnatal day-7 pups, and adult (8-week-old) mice.

Protocol compiled by: Caleb Heffner and Stephen Murray, Ph.D.

Reagents:

  • Slide fixative (0.2% Glutaraldehyde in PBS)
  • Detergent rinse (0.02% Igepal, 0.01% Sodium Deoxycholate, and 2mM MgCl2in 0.1M phosphate buffer [pH 7.5])
  • X-Gal staining solution (0.02% Igepal, 0.01% Sodium Deoxycholate, 5mM Potassium Ferricyanide, 5mM Potassium Ferrocyanide, and 2mM MgCl2 diluted in 0.1M phosphate buffer [pH 7.5])*

*Prepare the staining solution ahead of time, and store at room temp in the dark.   Add 1mg/ml X-Gal in N,N-dimethylformamide directly prior to use.

  • Post-stain fixative (4% Paraformaldehyde [PFA] in PBS)
  • Nuclear Fast Red (Vector Laboratories)
  • Permount (Fisher Chemical)
  • OCT Compound (Sakura)

Procedures:

15.5 dpc Embryos:

  1. Euthanize female by accepted ACUC protocol. Dissect out uterus, and remove all extra-embryonic tissues in cold PBS.
  2. Freeze embryos individually in OCT on dry ice.
  3. Section frozen blocks at 10 microns, and store cut slides at -80°C until staining.
  4. Immediately prior to staining, fix slides in slide fixative in PBS for 10 minutes on ice.
  5. Rinse in PBS, wash in PBS 10 min, wash in detergent rinse 10 min, immerse in 1mg/ml X-gal staining solution overnight at 37°C in the dark.
  6. Post-fix slides in 4% PFA for 10 min.
  7. Rinse in PBS, 10 min in PBS, wash 2 x 5 minutes in distilled water, counter-stain with Nuclear Fast Red, rinse in distilled water, and wash in distilled water for 2 minutes.
  8. Dehydrate, mount with Permount, and coverslip.

Postnatal Day 7 and Adult (8 weeks):

  1. Euthanize mouse by accepted ACUC protocols.
  2. Dissect desired tissues and freeze immediately in OCT on dry ice.
  3. Section, stain and mount slides in the same manner as described for 15.5 dpc embryos above.