While the timing of the various steps in this protocol are probably not critical, I tend to prefer to process the tissue all at once to ensure that RNA and/or proteins do not get degraded.
20% Paraformaldehyde/4% Paraformaldehyde-PBS
200 g paraformaldehyde
1 ml 10N NaOH
up to 1 liter with Q, heat to 65° to dissolve, aliquote and store at -20°.
Mix 100 ml 20% Paraformaldehyde with 50 ml 10X PBS and bring up to 500 ml with Q
filter, and store at 4° for up to 2 weeks
30% sucrose 150 g sucrose
up to 500 ml with 1X PBS
filter sterilize and store at room temperature
Dissect and fix the tissue in fresh 4% paraformaldehyde on ice for 5-10 minutes.
Wash for 5 minutes in 1X PBS and repeat.
Transfer to 30% sucrose until the tissue sinks (5-10 minutes depending on the size of the tissue).
Transfer through a 1:1 mixture of OCT:sucrose and then into OCT.
Place the tissue in the cryomold, overlay with OCT, orient and freeze quickly on dry ice.
Once the tissue is in the mold with OCT it should be oriented and frozen quickly because a film can form on the top of the mold (where the OCT is exposed to air) and make moving the tissue difficult.
If the size of the mold is small enough place each block into an eppendorf tube and store at -80°C.
Cut 20 micron sections and place on silinized superfrost slides (Protocol S.6).
For best results, proceed immediately with immunohistochemical staining.