Table 2. Troubleshooting the cryostat procedure.

FAULT	CAUSES
Cracks appear in frozen tissue	Freezing too rapidly or specimen is overly large
Specimen detaches from microtome stub	Insufficient embedding medium, or excessively vigorous block facing
Tissue advances but will not section	Knife is loose in its holder, specimen stub is loose in its mount, incorrect knife angle, anti-roll plate extending too far or tissue is unfrozen
Sections buckle or roll	Anti-roll plate has insufficient clearance or is incorrectly aligned, section thickness is too thin or knife is dull
Tears in section	Specimen is over frozen, knife edge is damaged or unclean
Sections thaw when cut	Incorrect cabinet or knife temperature
Frost appears on knife	Cryostat lid has been open too long
Section adhere to anti-roll plate	Insufficient anti-roll plate clearance, tissue or grease stuck to anti-roll plate, incorrect cabinet or knife temperature
Sections skew to one side	Debris on knife edge, nick in the knife, blunt knife edge, or a damaged anti-roll plate
Section detaches from slide	Fixed tissue sectioned, no slide adhesive used, fatty specimen, excessively vigorous handling
Section shows horizontal splits	Specimen was too cold when sectioned

Table 3. A selection of freeze-substitution agents.

Solvent	Substitution Temperature (°C)
Ethanol	-117
Diethyl ether	-116
Propylene oxide	-112
Acetone	- 95
Methanol	- 94
Acrolein	- 87
Ethanol-Mercuric Chloride	- 70
Acetone - OsO4	- 70