MDCK cells were cultured in Transwell cell culture inserts with a 0.4 μm pore polyester membrane coated with collagen for polarization. Cells were then fixed with 2% GA in 0.1 M NaCac buffer supplemented with 2 mM CaCl2. After 30 min fixation, cells were washed and osmicated with 1% reduced osmium prior to dehydration and flat embedding into epoxy resin. Resin was polymerized at 60°C overnight, and a pyramid was trimmed in the direction enabling cell cross-sections. The 60 to 70 nm sections were picked up on Pioloform-coated, single slot grids, and poststained with uranyl acetate and lead citrate prior to imaging with a JEM 1400 microscope equipped with an Orius SC 1000B CCD camera. For electron tomography, dual axis tilt series, ±62 degrees at one-degree intervals, were acquired from 230 nm-thick cross-sections using a Tecnai FEG 20 microscope operated at 200 kV. Images were collected at a nominal magnification of 7800×, providing a pixel size of 2.8 nm. Alignments and reconstructions were performed using the IMOD software (https://bio3d.colorado.edu/imod/). For volume EM, the images were acquired using a FEG-SEM Quanta 250 equipped with a 3View-system (Gatan, Inc). The image acquisition was performed with a pixel size of 5.5 nm, cutting depth of 50 nm, beam voltage of 2.5 kV, spot size of 3, and Torr pressure of 0.2. Segmentation and visualization were performed using Microscopy Image Browser (Belevich et al., 2016) and Amira softwares, respectively.

2023