Translocation of FC-5.01/CD63 complexes from the cell surface to early endosomes, lysosomes, and MIICs of immature DCs. Double labeling to detect internalized FC-5.01 Mab and EEA1, LAMP-2, and MHC class II molecules was performed. Immature DCs were incubated with 10 μg/mL FC-5.01 Mab for one hour on ice. After washing to remove unbound FC-5.01, cells were further incubated in culture medium at 37°C for the indicated periods of time. FC-5.01 was stained with Cy5-GAM (green in EEA1 panel) or Cy3-RAM (red in LAMP-2 and MHC class II panels). Cells were then incubated with rabbit anti-EEA1 (red; secondary antibody Cy3-GAR), goat anti-LAMP-2 (green; secondary antibody Cy5-RAG), or anti-HLADR, -DP, -DQ FITC-labeled (green). Colocalization is visualized by yellow staining. Images are representative of at least 4 similar experiments. Objective lenses: Plan-Neofluar 100 ×/1.3 NA, oil; digital zoom: 2.