Quantification of CLIC/GEEC Carriers

In transfected CAV1−/− MEFs, HRP uptake (10 µg/ml) was performed at 37°C for 2 min, and after brief washing with DMEM containing 1% BSA, diaminobenzidine (DAB) (10 mg/ml) reaction was performed on the live cell as previously described in [29] (link). Fixation, embedding, and sectioning were performed as follows. MEFs were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). Cells were post-fixed with 1% osmium tetroxide for 1 h at room temperature and serially dehydrated with ethanol. Cells were embedded in increasing ratios of LX-112 resin∶ethanol to 100% resin, and polymerized overnight at 60°C. Ultrathin (60 nm) sections were cut on a Leica UC6 microtome and imaged on a JEOL1011 electron microscope at 80 kV. Quantifications were performed as follows: the perimeters of approximately 16 cells (per experimental condition) were imaged and the number of CLICs/GEEC carriers from each cell was quantified and averaged across all 16 cells. The average number of CLICs/GEEC carriers per cell was generated from two separate repeats of the same experimental conditions.