Purine-like TLR7 agonist SM360320 colocalizes with imiquimod in the MIIC. pDCs, precultured overnight with IL-3, were stimulated with SM-TAMRA (20 μm) or with SM-AF488 (3 μm), alone or together with SM-TAMRA (20 μm) or R837-TAMRA (20 μm), for 90 minutes. Cells were fixed, permeabilized, and stained with anti–HLA-DR-APC or purified rabbit polyclonal anti-LAMP1 Ab followed by anti–rabbit Ab labeled with AF647. 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.