TABLE 2.

The protocol of freeze-substitution and resin embedding.

Step	Medium	Temperature	Time
1	0.5% OsO4 in acetone	−90°C	2 h
2	-	−90°C → −20°C, 2 degrees/h	35 h
3	Wash with acetone	−20°C	3 × 10 min
4	0.25% tannic acid in acetone	−20°C → −10°C, 4 degrees/h	2.5 h
5	Wash with acetone	−10°C	3 × 10 min
6	0.25% OsO4 in acetone	−10°C → 0°C, 4 degrees/h	2.5 h
	Epon resin embedding
7	25% resin in acetone	0°C → 20°C, 5 degrees/h	4 h
8	50% resin in acetone	RT	2 h
9	75% resin in acetone	RT	2 h
10	100% resin	RT	2 h
11	100% resin	RT	2 h
12	Polymerization
	100% resin	40°C	24 h
	-	65°C	72 h

RT, room temperature.