Fig. 7. Polarized MDCK cells expressing F_c38-63 were grown on permeable supports. The basolateral surface of the cells was incubated with anti-F_c receptor antibodies (A) for 30 minutes at 4°C. The cells were then washed and shifted to 37°C for 2 minutes. After 2 minutes of incubation, the cells were shifted to citrate buffer, pH 1.5, for 5 minutes at 4°C to elute surface-bound antibodies. The cells were then rinsed in PBS and fixed. Following permeabilization, the cells were incubated with antibodies specific for caveolin 1 (B). The cells were again washed and incubated with anti-rat IgG conjugated to Alexa Fluor-594 (A) and anti-rabbit IgG conjugated to Alexa Fluor-488 (B). The merged image (C) illustrates the overlap between the chimera and caveolin. The xy-slice in each confocal image is approximately 1 µm above the basal membrane of the cell. Similar analyses have quantified the colocalization of F_c38-63 with clathrin, transferrin, Ctx-B or EEA1. The bar graph in D shows the percentage of the total number of pixels resulting from F_c38-63 staining that colocalized with these various endocytic markers on the cell surface (0 Min.), and at 2 and 5 minutes post-internalization. This quantification reflects the average result from at least 25 cells from two independent experiments. Bar, 1 µm.