Fixation methods for electron microscopy of human and other liver

Table 1 Dehydration and embedding for TEM and SEM

TEM
Ethanol 70%	15 min	20°C
Ethanol 90%	15 min	20°C
Ethanol 100%	30 min	20°C
Ethanol 100%	30 min	20°C
Ethanol 100%/Epon 1/1	Overnight	20°C
Epon	1 h	20°C
Epon 1 (overnight)	BEEM capsules on top	20°C
Epon	Fill BEEM capsules	20°C
Oven	67 h	40-60°C
SEM
Ethanol 70%	15 min	20°C
Ethanol 90%	15 min	20°C
Ethanol 100%	30 min	20°C
Ethanol 100%	30 min	20°C
Ethanol 100%	Overnight	20°C
Freeze and fracture	Liquid nitrogen	-196°C
Ethanol 100%	30 min	20°C
Critical point drying, phase 1	CO2/ethanol	16°C
Phase 2 or HDMS drying (see below)	80 bar	35°C
Sputter coating	Pt/Au

Cutting of ultrathin sections on an ultramicrotome, contrast staining with lead and uranyl salts. TEM: Transmission electron microscopy; SEM: Scanning electron microscopy.