Whole mount β-galactosidase staining

The Jackson Laboratory Cre Repository uses whole-mount β-galactosidase staining for 10.5 dpc embryos.

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

Reagents:

Whole-mount fixative(0.2% Glutaraldehyde, 5mM EGTA, and 2mM MgCl2 in 0.1M phosphate buffer [pH 7.5])

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)

Procedure:

1 Euthanize female by accepted ACUC protocol. Dissect out uterus, and remove all extra-embryonic tissues in PBS.

2 Fix embryos for 40 min in whole-mount fixative on ice.

3 Rinse 3 x 15 min in detergent rinse.

4 Stain by immersion in 1mg/ml X-gal staining solution overnight at 37°C in the dark.

5 Rinse embryos in PBS, and postfix/ store in 4% PFA in PBS at 4°C.

6 Image, dehydrate and embed embryos in paraffin blocks for sectioning. Section embryos at 5 µm, de-wax, re-hydrate, counter-stain with Nuclear Fast Red, dehydrate and mount slides with Permount.