Fig. 3. MDCK cells expressing Fc38-63 were incubated in serum-free DMEM that lacked (A-C) or contained (D-F) 10 mM methyl--cyclodextrin for 1 hour. Cells were then fixed, permeabilized and incubated with rat anti-F_c-receptor antibody and with a rabbit antibody directed against furin. After washing, cells were incubated with anti-rat IgG conjugated to Alexa Fluor-594 and anti-rabbit IgG conjugated to Alexa Fluor-488. Alternatively, after a 1-hour incubation in 10 mM methyl--cyclodextrin, the cells were washed with DMEM containing 5% fetal bovine serum and incubated for 20 minutes (G-I) or 60 minutes (J-L) with 10 mM methyl--cyclodextrin loaded with cholesterol and processed as described above. The upper panel in each confocal image corresponds to an xy-slice near the middle of the cells, whereas the lower panel corresponds to an xz-slice. Black bars mark the position of the basal membrane. Bars, 10 µm.