Fluorescently labeled R848 and R837 localize to LAMP1⁺HLA-DR⁺ endosomes in pDCs. pDCs, precultured overnight with IL-3, were stimulated with R848-TAMRA (20 μm), R837-TAMRA (50 μm), R837-AF488 (20 μm) alone or together with R837-TAMRA (50 μm) for 90 minutes. Cells were fixed, permeabilized, and stained with purified rabbit polyclonal anti-LAMP1 Ab followed by anti–rabbit Ab labeled with AF488 (top and upper middle) or AF568 (lower middle) and anti–HLA-DR-APC. Images were acquired with a ZEISS LSM 710 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.