350 ml 0.1 M PB solution (pH 7.35)

150 ml ethylene glycol

100 μg sodium azide

150 g sucrose

Final cryoprotectant concentration will be: 30% ethylene glycol, 30% sucrose, 0.00002% sodium azide, in 0.1 M PB (pH 7.35).  Prepare this solution under the hood.

1. Pour 250 ml of 0.1 M PB into 1 L flask.
2. Pour 150 ml of ethylene glycol into the flask.
3. Add 100 μg sodium azide.
4. Stir for 30 min
5. Add 150 g of sucrose
6. Stir for 2 hr
7. Bring volume up to 500 ml with 0.1 M PB
8. Store cryoprotectant solution in refrigerator (+4ºC)