Table 1.

Freeze-substitution programs for AFS 1 and AFS 2

AFS 1	AFS 2
−90°C for 30–36°C	−90°C for 30–36°C
5°/h to −50°C	5°/h to −50°C
−50°C for 2 h	Pause
5°/h to −30°C	Unpause after acetone wash
−30°C for 72 h	5°/h to −30°C
Stop the program and initiate another program after plastic embedding	Pause
−30°C for 0 h	Unpause after plastic embedding
10°C/h to −20°C	10°C/h to −20°C
−20°C for 24 h	−20°C for 24 h

Note that two programs must be used for AFS 1.