1.Embed fresh tissues carefully in OCT in plastic mold, being careful not to trap air bubbles surrounding the tissue. Freeze tissue by setting mold on top of liquid nitrogen until 70-80% of the block turns white. Then, put block on top of dry ice. Frozen blocks may be stored at -80C for long-term storage.
2.For cutting step, mount the frozen block on the cryostat holder. Never, at any point, let the tissue warm up to temperatures above -15C.
3.Allow frozen blocks to equilibrate in the cryostat chamber for about 5 minutes. Cut 6-8 um sections. The best sections are usually obtained when the block temperature is around minus 18 to minus 20C.
4.Let the sections dry for at least 30 minutes at room temperature. Note: Upon drying, tissues can be stored at 4C for a few days; for longer-term storage up to a few months, store at -80C.
5.Fix the sections by immersing in acetone jar (or other appropriate fixatives) for 1-2 minutes at room temperature, and let air-dry. Carefully draw boundaries of sections using a PAP Pen and let it dry for a few minutes.