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:
- Euthanize female by accepted ACUC protocol. Dissect out uterus, and remove all extra-embryonic tissues in cold PBS.
- Freeze embryos individually in OCT on dry ice.
- Section frozen blocks at 10 microns, and store cut slides at -80°C until staining.
- Immediately prior to staining, fix slides in slide fixative in PBS for 10 minutes on ice.
- 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.
- Post-fix slides in 4% PFA for 10 min.
- 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.
- Dehydrate, mount with Permount, and coverslip.
Postnatal Day 7 and Adult (8 weeks):
- Euthanize mouse by accepted ACUC protocols.
- Dissect desired tissues and freeze immediately in OCT on dry ice.
- Section, stain and mount slides in the same manner as described for 15.5 dpc embryos above.
