Table 2.

Timeline of Tissue Processing.

Step	Procedure	Solution	Duration
1	Subject perfused	4% paraformaldehyde or Krebs buffer, and then 4% paraformaldehyde (two-stage perfusion)
2	Brain postfixed	4% paraformaldehyde	24 hr
3	Cryoprotection stage 1	10% glycerol + 2% DMSO	1–3 days
4	Cryoprotection stage 2	20% glycerol + 2% DMSO	3–5 days
5	Flash freeze the cryoprotected block and hold at −75C for 1 hr	−75C isopentane	Begins “total time frozen”
6	Move frozen block to −80C storage		Until next step (days to years)
7	Cut frozen block cut on freezing sliding microtome and rapidly thaw sections on blade	Collect thawed tissue sections in 15% glycerol	Store at 4C overnight
8	Freeze sections at −80C	Same 15% glycerol	Until next step (days to years)
9	Thaw all sections rapidly as needed for batch-processed histochemical study		Ends “total time frozen”

The steps used to obtain, cryoprotect, cut into sections, and store tissue sections before histochemical processing are shown in order. The total time frozen encompasses the time between flash freezing the cryoprotected block (step #5) and thawing tissue sections on the first day of histochemical processing (step #9). All solutions are prepared in 0.1-M phosphate buffer, pH 7.4.