Cells were seeded on sapphire disks for 24 h and incubated with BSA5 for 3 h before HPF. 10 min before HPF, cells were washed with full DMEM. The sapphire disks and cells were transferred into sample holders and inserted into a Leica EM ICE (Leica Microsystems) and frozen. After freezing, the samples were stored in LN2. Samples were freeze substituted with chemical fixative and postfixed in 2% OsO4. Cells were further processed in EPON in increasing concentrations and finally embedded in 100% EPON, polymerized for 3 d at 60°C.
Resin embedded cells were prepared for thin sectioning by removal of the sapphire disk and trimming of the stub to a rectangle of ∼0.5 by 1 µm. 60−70 nm Sections were made on a Leica Ultracut S (Leica microsystems) using a DiATOME Ultra Diamond Knife 45°. Sections were deposited on Formvar- and carbon-coated copper grids and poststained using uranyl and lead citrate in a Leica EM AC20 (Leica microsystems). Samples were imaged on a Tecnai T12 (FEI Tecnai) transmission electron microscope (TEM) using SerialEM software (Mastronarde, 2018 ). Image tileset stitching was done with Etomo EM processing software (Mastronarde and Held, 2017 (link)).
Resin embedded cells were prepared for thin sectioning by removal of the sapphire disk and trimming of the stub to a rectangle of ∼0.5 by 1 µm. 60−70 nm Sections were made on a Leica Ultracut S (Leica microsystems) using a DiATOME Ultra Diamond Knife 45°. Sections were deposited on Formvar- and carbon-coated copper grids and poststained using uranyl and lead citrate in a Leica EM AC20 (Leica microsystems). Samples were imaged on a Tecnai T12 (FEI Tecnai) transmission electron microscope (TEM) using SerialEM software (Mastronarde, 2018 ). Image tileset stitching was done with Etomo EM processing software (Mastronarde and Held, 2017 (link)).
