Colocalization of hOSCAR-internalized material with Lamp-1 and HLA-DR after 20 minutes of endocytosis. Mono-DCs were allowed to adhere onto poly-l-Lysine slides and incubated with hOSCAR-specific F(ab′)2 in the presence of 2% human serum. These primary mAbs then were cross-linked with Alexa 594–conjugated GAM F(ab′)2, and the cells were moved to 37° C (B-CE-F) to allow endocytosis or kept on ice (AD). After 20 minutes, the cells were moved to 4° C, fixed, and permeabilized to allow labeling of intracellular markers (A-C: anti–Lamp-1–FITC; D-F: anti–HLA-DR biotin and Alexa 488–conjugated streptavidin). To analyze the relative localization of the material internalized by hOSCAR (in red) and the intracellular markers (in green), the pictures were merged, and double-labeled areas appeared in yellow. Upon 20 minutes endocytosis, the intracellular compartments accessed by hOSCAR-internalized material were Lamp-1 (B-C) and HLA-DR positive (E-F). Bars: 10 μm. The data are representative of 2 experiments.
