Triggering DC-SIGN neck domain leads to a prolonged localization of the antigen in early endosomes and MHC class I compartments. (A) Immature DCs were incubated with anti-neck antibodies H200 or DCN46 (green) at 4°C, washed, and shifted at 37°C for the indicated time points. Cells (except for t = 0 samples) were acid-strip treated to remove extracellular membrane-bound antibodies, fixed, permeabilized, stained with anti-EEA-1 antibody (red), and analyzed by confocal microscopy. Representative images from at least 3 different experiments are shown. Scale bars represent 10 μm. Immature DCs were incubated with antineck antibody H200 (green) and OVA-Alexa647 (blue) at 4°C, and then treated as in panel A. After acid-strip, fixation, and permeabilization, the samples were stained with anti-EEA-1 (B,D) or anti-MHC-I (C,E) antibodies (red) and analyzed by confocal microscopy. Representative images from 2 independent experiments are shown. Scale bars represent 10 μm.