Imidazoquinolines localize to LAMP1⁺CD63⁺ endosomes but not to TfR⁺EEA-1⁺ endosomes in pDCs. Human pDCs, precultured overnight with IL-3, were stimulated for 90 minutes with (A) R848-TAMRA (20 μm), R837-TAMRA (50 μm), CpG-B–TAMRA (10 μm), or CpG-A–TAMRA (10 μm); (B) R837-TAMRA (50 μm), CpG-B–TAMRA (10 μm), CpG-A–TAMRA (10 μm), or R837-TAMRA (50 μm) together with CpG-B–FITC (10 μm); (C) R837-TAMRA (50 μm), CpG-B–TAMRA (10 μm), CpG-A–TAMRA (10 μm) alone or together with R837-AF488 (20 μm); (D) R837-AF488 (20 μm), CpG-B–FITC (10 μm), R848-TAMRA (20 μm), or CpG-B–TAMRA (10 μm). Cells were fixed, permeabilized, and stained with anti–LAMP-1-FITC Ab (A), anti–CD63-FITC Ab (B), purified mouse mAb anti-TfR followed by anti–mouse Ab labeled with AF488 (C), or purified rabbit mAb anti-EEA1 followed by anti–rabbit Ab labeled with AF568 (D, top and upper middle) or AF488 (D, lower middle and bottom). Images were acquired with a Bio-Rad TE2000-U (A-B) or a ZEISS LSM 710 (C-D) confocal microscope and an oil-immersion objective (63×/1.4 NA), with the pinhole set for a section thickness of 0.8 μm (pinhole set to 1 airy unit in each channel). Images were acquired sequentially with separate laser excitations to avoid cross talks between different fluorophores and processed with Zen2008 software. Images show single cells from 1 representative donor of 3.