Orius sc 1000b camera

U2OS cells were loaded with 400 μM oleic acid overnight and fixed with 4% paraformaldehyde in MES-sucrose buffer (10 mM MES/138 mM KCl/3 mM MgCl₂/2 mM EGTA/11.14% sucrose) for 2 h. The cells were then permeabilized with 0.01% saponin in 0.1% BSA/0.1 M Na-PO₄ pH 7.4 for 8 min, labelled with the rabbit anti-NMIIa antibody (Covance, PRB-440 P 1:50) for 2 h followed by the nano-gold-conjugated anti-rabbit Fab-fragments for 1 h (Nanoprobes, #2004, 1:60), post-fixed in 10% glutaraldehyde, and quenched in 50 mM glycine. The nano-gold particles were intensified using the HQ SILVER Enhancement kit according to the manufacturer's instructions (Nanoprobes) followed by gold toning in subsequent incubations in 2% NaAcetate, HAuCl₄ and 0.3% Na₂S₂O₃·5H₂O. The cells were treated with 1% OsO₄ supplemented with 15 mg ml⁻¹ K₄[Fe(CN)]₆ and flat-embedded in Epon as described earlier39 (link). Ultrathin sections were cut using Leica UCT microtome, picked on pioloform coated single slot grids, post-stained with uranyl acetate and lead citrate, and observed with the Jeol JEM-1400 microscope at 80 kV. Images were acquired with the Gatan Orius SC 1000B camera.

Single-axis tilt series were recorded on a JEM 2100 or a JEM 1400 transmission electron microscope (JEOL) operated at 200 or 120 kV, respectively. The tomography plug-in of the Digital Micrograph software (Gatan) was used to acquire images automatically every 2° over a ±60° range using an Ultrascan 2K × 2K CCD camera (Gatan) at a pixel size of 1.01 nm (JEM 2100) or a Gatan Orius SC1000B camera at a pixel size of 0.64 nm (JEM 1400) at a magnification of ×10,000. Tomograms were reconstructed with IMOD by the simultaneous iterative reconstruction technique (SIRT). The nominal resolution in our tomograms is estimated at about 4 nm according to the Crowther criterion^{62 (link)}.