Fix cells/tissue in 4% paraformaldehyde in 0.25 M Hepes pH 7.4 for 1 hour at room temperature.
Replace fixative with 8% paraformaldehyde in 0.25 M Hepes pH 7.4 and leave overnight @ 4°C.
Wash 3 times in PBS. In case of cell monolayers, scrape the cells with a rubber policeman and pellet them @ 5,000 - 10,000 RPM for 5 min @ RT.
Embedding in Gelatin
Melt gelatin (10% in PBS) by warming it up in a waterbath at 45°C.
Resuspend pellet in 0.5 ml of gelatin. Spin immediately @ RT, then put on ice.
Let gelatin set completely for 1 h on ice.
Using a wooden stick, loosen the block of gelatin from the tube and transfer to a dish on ice.
Using a razorblade or scalpel, cut the tip of the pellet into small pieces.
Infiltration with Sucrose
Transfer pieces into an Eppendorf tube containing 2.3 M sucrose in PBS.
Gently swirl the bottle and leave overnight at 4°C.
Freezing Down Samples
Transfer pieces of pellet/tissue onto plastic nail-holder making sure to cover them completely with sucrose. Dissect them into smaller pieces using a razorblade.
Transfer the small pieces onto aluminum nails/studs, leaving just enough sucrose to “glue” them to the nail.
Immediately plunge the nail into liquid nitrogen using the special orange forceps.
Transfer the nails into a cryotube (Nunc) and store in liquid nitrogen.
Return to:
U of M Home
