Leica em afs

Cells were transferred into sample holders with a depth of 200 μm and a diameter of 1.2 mm with a small amount of culture medium. Cryofixation was performed by means of a Leica EM PACT high pressure freezer (Leica Mikrosysteme GmbH, Vienna, Austria) within milliseconds at about 2,000 bar. Eppendorf tubes with the sample holders containing the fixed specimens were filled with a precooled mixture made of 2% osmium tetroxide and 0.05% uranyl acetate in acetone and placed in a Leica EM AFS (Vienna, Austria) for freeze substitution. The AFS was programmed as follows: 60 h at −80 °C, followed by a temperature increase at a rate of 10 °C per hour for 5 h to −30 °C, 4 h at −30 °C, and another temperature increase at a rate of 2.5 °C per hour for 20 h to 20 °C, and finally 10 h at 20 °C. After freeze-substitution, the samples were rinsed three or four times in anhydrous acetone followed by three times washing for 10 min each in propylene oxide before embedding in Agar Low Viscosity Resin (ALVR; Agar scientific, Stansted, UK). The embedding was done with a propylene oxide:ALVR ratio of 3:1, 1:1, and finally 1:3. Next, the samples were placed for 4 d in a desiccator for polymerization and finally for 24 h in an oven at a temperature of about 60 °C.