HPF & FS
High-pressure freezing and freeze-substitution.
1) Tissue as NMJ.
2) Monolayer cultured cells.
1) Place tissue/cells in specimen carrier (200 µm depth) filled with yeast paste.
2) Vitrificate using HPF machine.
3) Put the frozen specimens in cold pure acetone (with tannic acid or osmium) at -90°C for several hours – days.
4) Change the temperature automatically at -60°C, –30°C and 4°C.
5) Wash the specimen with pure acetone at 4°C.
6) Infiltrate with a range of resin & acetone mixtures.
7) Polymerise in resin at 60°C for 2 days.