Whole Mount X-Gal Staining

Solutions:

Rinse Buffer 1X PBS 100 ml 10X PBS 2mM MgCl2 2 ml 1.0 M MgCl2 0.01% sodium deoxycholate (Sigma D-5670) 100 mg 0.02% NP-40 200  l 1 L Ferro For 10X, 1.05 g in 50 ml Rinse Buffer  Store at RT, wrapped in foil

Ferri For 10X, 0.82 g in 50 ml Rinse Buffer  Store at RT, wrapped in foil

***weigh and mix ferro and ferri solutions in hood!!

X-gal 25 mg/ml stock in DMF, stored at -20C (500 l aliquots)  dilute 1:25, so final concentration is 1 mg/ml X-gal

Staining solution Per vial 5 mM Ferro 20 l of 10X Ferro 5 mM Ferri 20 l of 10 X Ferri 1 mg/ml X-gal 800 l of 25 mg/ml X-gal Rinse Buffer 20ml

1. Prepare fresh 4% Paraformaldehyde/PBS, pH 7.0-7.5 2. Anaesthetize mice with isoflurane and sacrifice by cervical dislocation. 3. Collect organs of interest and fix 2 hours in 4% PFA/PBS 4. Rinse 3x with Rinse Buffer, for 15 minutes each at 4C. 5. Add 20 ml staining solution per vial and stain overnight at 37C 6. Next day, replace staining solution with 10% formalin to post-fix. 7. Change formalin after 24 hours. 8. Change to 70% EtOH after 24 hours.