X-GAL STAINING OF WHOLE EMBRYOS

Josh Hall

Finger Lab 11/98

REAGENTS

• Wash Buffer
• 1x PBS (1:10, 10x PBS)
• 5 mM EGTA (1:100, 500 mM EGTA)
• 2 mM MgCl₂ (1:500, 1 M MgCl₂)
• 0.01% deoxycholate (10 mg/100 ml solution)
• 0.02% Nonidet P-40 (20 µl/100 ml solution ~ 1:5000)

• Staining buffer
• Same as wash buffer with:
• 1 mg/ml X-gal (1:50, 50 mg/ml X-gal - store at -20°C in dark)
• 10 mM Potassium ferrocyanide (1:50, 500 mM stock - store in dark)
• 10 mM Potassium ferricyanide (1:50, 500 mM stock - store in dark)

Also: PBS, 4% Paraformaldehyde in PBS

PROCEDURE

1. Collect embryos into cold PBS, place in baked 60 mm glass dish

2. Wash twice with PBS briefly

3. Fix 2 hours at room temp in 4% PFA in PBS (5-10 ml for 10 embryos)

4. Wash 3x15 minutes in Wash Buffer (5-10 ml/wash)

5. Place in staining buffer and incubate at 37°C 1-24 hours. If ISH will be done after X-gal, stain 3-4 hours.

6. Wash embryos in PBS and refix 2 hours at room temp (or overnight at 4°C) in 4% PFA in PBS.

7. Continue with ISH protocol or store embryos at 4°C. Can store a few days in PBS, for prolonged storage place in 70% ethanol.

REFERENCES

1. Gossler, A. and J. Zachgo, Gene and enhancer trap screens in ES cell chimeras, in Gene Targeting: A practical approach, A.L. Joyner, Editor. 1993, IRL Press: Oxford. p. 208-209.

2. Tajbakhsh, S. and D. Houzelstein, In situ hybridization and beta-galactosidase: a powerful combination for analysing transgenic mice. Trends Genet., 1995. 11(2): p. 42.