BOT/MBIOL/ZOOL 5364

CRYOPROTECTANT FOR PROPANE JET FREEZING (Ding et al. 1991)

To make 20 ml of buffer for propane jet cryoprotection:

                                Chemical                                   Amount needed                           FW

                     20 mM               MES                              0.07808 g                              195.2

                      2 mM               CaCl₂                             0.00588 g                              147.02

                      2 mM               KCl                                0.00298 g                                74.557

                      0.2 M               sucrose                         1.3692                                   342.30

Adjust pH to 5.5 using KOH.

Procedure:

Incubate material in the buffer for 5 to 30 minutes before freezing.  Ideally, the tissue should be as small as possible.  (See requirements of propane jet freezer.)

Reference:

Ding, B., Turgeon, R. and Parthasarathy, M. V. (1991)  Routine cryofixation of plant tissue by propane jet freezing for freeze substitution.  Journal of Electron Microscope Technique 19: 107-117.