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.
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