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Fig. 5. Internalized ALCAM/CD166 colocalizes primarily with clathrin. A2774 cells were stained either with I/F8 scFv plus anti-FLAG mAb (A,D) or with J4-81 mAb (B,E), allowed to internalize for 40 minutes at 37°C, acid-washed to remove the surface-bound antibody, fixed, permeabilized and stained with fluorochrome-labeled secondary antibody. As the colocalization with other molecules was investigated, the secondary antibody free valences were saturated by a 30-minute incubation with mouse IgG, followed by a second round of PFA fixation, and finally the cells were incubated with FITC-labeled anti-clathrin or with anti-caveolin antibodies. Anti-caveolin was then detected with RITC-labeled goat anti-mouse IgM antibody. As colocalizing positive controls, transferrin-RITC (TF, panel C) and cholera toxin B-FITC (CT B, panel F) were allowed to internalize for 10 and 40 minutes, respectively, at 37°C. Cells were then acid-washed, fixed, permeabilized and processed for staining with anti-clathrin and anti-caveolin antibodies. Double immunofluorescence was visualized by confocal microscopy. Single-color immunofluorescence images are shown in the small panels and the corresponding merged images are shown in the large panels. Original magnification, 600 x.
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