Targeting to DEC205 results in late endosomal localization, and CD40 and MR antibodies are largely excluded from late endosomes. (A-C) BDCA1⁺ DCs or (D-E) MoDCs were continuously incubated with 1 μg/mL fluorescently labeled (A,D) anti-DEC205, (B,E) anti-MR, or (C,F) anti-CD40 antibodies for 6 hours at 37°C. In parallel, DCs were incubated with antibodies in the presence of protease and acidification inhibitors leupeptin and NH₄Cl (inh). DCs were seeded on coverslips and fixed, followed by staining for the cell surface (HLA-DR) and permeabilization and labeling of early endosomes (EEA1) or late endosomes (Lamp1). Arrows indicate areas of overlap. Images were acquired on a Leica SP5 confocal microscope, 100× oil objective (NA, 1.47), zoom 7. All immunofluorescence experiments were repeated in ≥ 5 independent donors. Scale bar is 5 μm.