Megaview g2 ccd camera

MDCK II cells were cultured on Transwell filters (0.4 $\mathrm{\mu }\mathrm{m}$ pore, polycarbonate; #3412; Corning) for 5 days. After rinsing once with 0.1 M phosphate buffer (pH 7.4), the cells were fixed with 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) overnight at 4 $°\mathrm{C}$ . After washing three times with 0.1 M phosphate buffer (pH 7.4), the cells were cryoprotected with 30% glycerol in 0.1 M phosphate buffer (pH 7.4) for 30minat RT. After excision of the filters with scalpels, the cells were scraped off from the filter and mounted on gold stubs. After removal of the excess buffer, the samples were snap-frozen in liquid nitrogen. The frozen samples were transferred to the freeze-fracture system (BAF-060; Bal-tec). The samples were fractured at −110 $°\mathrm{C}$ and coated with a thin layer ( $\sim$ 2 nm) of platinum at a $45°$ angle, followed by a coating with a thin layer ( $\sim$ 20 nm) of carbon at a 90° angle. After retrieval of the samples, the samples were coated with collodion and cleaned with domestic bleach. The replicas were washed three times with water and collected on 200-mesh formvar-coated copper grids. Samples were observed with a JEM1011 transmission EM (JEOL) at 100-kV accelerating voltage. Images were captured with a MegaViewG2 CCD camera using iTEM software (Olympus Soft Imaging Solutions). The apical surface was identified based on the appearance of microvilli.