Afs2 freeze substitution device

Exponentially growing cells were centrifuged (3,000g, 5 min) and the pellet was loaded on aluminium discs with depth of 100 mm (Engineering Office M. Wohlwend GmbH, Switzerland) and covered with a flat disc. The sandwiched sample was frozen in a HPM010 high-pressure freezing machine (Bal-Tec, Liechtenstein). Cells were subsequently freeze-substituted in a AFS2 freeze substitution device (Leica Microsystems, Austria) in anhydrous acetone containing 2% glutaraldehyde and 0.2% tannic acid osmium tetroxide for 3 days at −90 °C and then warmed up to −30 °C over 24 h. Samples were washed thrice with acetone, incubated for 1 h at room temperature with 2% osmium tetroxide, washed thrice with acetone and infiltrated for 5–7 days at room temperature in a series of increasing concentration of Epon in acetone. After polymerization at 60 °C, 60–80 nm sections were stained with uranyl acetate and lead citrate and examined in a Tecnai T12 electron microscope (FEI, Holland) operating at 120 kV, utilizing a 2k by 2k ES500W Erlangshen CCD camera (Gatan, UK).

The cells were centrifuged,
and the pellet was loaded on aluminum disks with a depth of 100 μm
(Engineering Office M. Wohlwend GmbH, Switzerland) and covered with
a flat disc. The sandwiched sample was frozen in an HPM010 high-pressure
freezing machine (Bal-Tec, Liechtenstein). The cells were subsequently
freeze-substituted in an AFS2 freeze substitution device (Leica Microsystems,
Austria) in anhydrous acetone containing 2% glutaraldehyde and 0.2%
tannic acid osmium tetroxide for 3 days at −90 °C and
then warmed up to −30 °C over 24 h. The samples were washed
three times with acetone, incubated for 1 h at room temperature with
2% osmium tetroxide, washed three times with acetone, and infiltrated
for 5–7 days at room temperature in a series of increasing
concentrations of Epon in acetone. After polymerization at 60 °C,
60–80 nm sections were stained with uranyl acetate and lead
citrate and examined in a Tecnai T12 electron microscope (FEI, Holland)
operating at 120 kV, utilizing a 2k × 2k ES500W Erlangshen CCD
camera (Gatan, U.K.).