Fig. 7. The surface distribution and steady-state localization of wild-type and mutant F_c1-37 constructs in polarized MDCK cells. Polarized MDCK cells transiently (A,C) or stably (B) expressing F_c1-37 (A and B) or F_c1-37S25A (C) were grown on Transwell filters. The intact cells were either incubated with the F_c-receptor-specific antibody for 1 hour at 4°C, washed and fixed in 3% paraformaldehyde (A) or the cells were fixed in 3% paraformaldehyde, permeabilized by incubation in PBST and incubated with the F_c-receptor-specific antibody (B,C). The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine (A-C) and phalloidin conjugated to FITC (B and C). Following washing, the distribution of fluorescently labeled proteins was visualized using a Zeiss LSM510 confocal microscope. The 0.5 µm xy image in each panel is either near the center (A,B) or near the apical surface (C) of the cells. The surface population of F_c1-37 could be detected in a longer exposure of the image in B. The black arrowhead next to each panel marks the position of the basal membrane in the xz image. Bars, 10 µm.