The Protocol of Cryosectioning

Cryosectioning is a procedure that involves freezing tissue sections that have been fixed in a chemical such as paraformaldehyde. After the section is sufficiently frozen, you can then cut it to the desired thickness.

Fixation

The tissue must first be dissected and then fixed, using a four percent solution of paraformaldehyde on ice for five to 10 minutes. Wash the section for five minutes in phosphate-buffered saline and repeat the rinse, then transfer the tissue to a 30 percent sucrose solution until it sinks.

Infiltration

Transfer the tissue through a 50:50 mixture of sucrose and OCT (a compound that gets its name from Optimal Cutting Temperature), and then into pure OCT.

Embedding

Place the section into a cryomold and cover with OCT. Freeze the section quickly on dry ice to avoid formation of a film.

Cutting and Staining

Once the section is frozen, you can cut sections of 20 microns in thickness and then stain the sample as quickly as possible for the desired test. Alternatively, store the section at -80 degrees Celsius.

References

• Harvard Medical School: Cryosectioning Protocol C7

Resources

• University of Alabama at Birmingham: Freezing Tissues for Cryosectioning