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.

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

30% sucrose 150 g sucrose

up to 500 ml with 1X PBS

• 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.