Em 902

COS7 were prepared for electron microscopy, as previously described (Wakabayashi et al., 2009 (link)). In brief, cells were fixed for 1 h in prewarmed 2% (vol/vol) glutaraldehyde, 2.5% (mol wt/vol) sucrose, 3 mM CaCl₂, and 100 mM Hepes-KOH, pH 7.4. After washes, cells were postfixed using reduced OsO₄ (1% [mol wt/vol] OsO₄, 10 mg/ml potassium ferrocyanide, 1.25% [mol wt/vol] sucrose, and 100 mM sodium cacodylate, pH 7.4) for 1 h on ice. Cells were then incubated in 2% (mol wt/vol) uranyl acetate for 30 min. After dehydration using 50, 70, 90, and 100% ethanol, samples were embedded in Epon (Fluka). Samples were observed under a transmission electron microscope (EM 902; Carl Zeiss) at an acceleration voltage of 80 kV, and micrographs were acquired using a camera (MegaView 3; Olympus).

Transmission electron microscopy assays were performed as previously described [54]. Briefly, cells or human biopsies were fixed for 1 h at 4°C in 1.6% glutaraldehyde in 0.1 mol/L phosphate buffer, pH 7.2, then washed and fixed again in aqueous 2% osmium tetroxide, dehydrated in ethanol, embedded in Epon (Serva, 21045.02), and processed for electron microscopy with a Zeiss EM 902 transmission electron microscope at 80 kV. Ultra-thin sections were cut and stained with uranyl acetate and lead citrate.

Cells were seeded on coated petri dishes and cultivated until confluency. Then, the cells were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate for 20 min on ice. The cells were rinsed with 0.1 M sodium cacodylate buffer containing 3 μM calcium chloride and incubated in a mixture of 1% OsO₄, 0.8% potassium ferrocyanide, 3 μM calcium chloride in 0.1 M sodium cacodylate for 30 min. Then, the cells were washed with double distilled water and stained with 2% uranyl acetate before incubating in increasing EtOH solutions (20%, 50%, 70%, 90%, 100%). Subsequently, the cells were infiltrated with a mixture of 50% EtOH and 50% Durcupan ACM resin (Fluka), and then incubated at least 3 × 2 h in 100% Durcupan and polymerized for at least 48 h at 60 °C in an oven. For TEM, ultrathin sections with a thickness of 70 nm were analyzed on a Zeiss EM902. For electron tomography, semi-thick sections with a thickness of 300–500 nm were poststained in 2% uranyl acetate and Sato lead. As fiducial markers, 20-nm sized gold nanoparticles were deposited on each side of the grid. For each reconstruction, a tilt series of images was recorded at 1-degree tilt increments on a FEI Titan 80–300 (CTWIN) IVEM/STEM. The electron microscopes were operated at 80 kV (Zeiss) and 300 kV (FEI).