(A) WT C57BL/6J mice were treated daily with IMQ (labeled as IMQ⁺) or control Vaseline cream (labeled as IMQ^–) application on back skin for 5 consecutive days. (B) Representative H&E-stained skin sections (total original magnification, ×400; scale bar: 40 μm) from A. (C–F) Epidermal cells freshly isolated from mouse trunk skin were stained with anti-MHC-II, CD45.2, EpCAM, Ly6C, langerin, CD80, and CD86 Ab and analyzed by flow cytometry. (C) Representative FACS analysis and the ratios of epidermal LCs (MHC-II⁺CD45.2⁺) (n = 30, 7 independent experiments). (D) Representative histograms of langerin, EpCAM, and Ly6C expression in LCs (dark gray filled, IMQ-untreated; black line, IMQ-treated; for langerin, n = 12, 3 independent experiments; for EpCAM, n = 16, 4 independent experiments; for Ly6C, n = 4, 1 independent experiment). (E and F) Representative FACS analysis, positive ratios, histogram, and MFI of CD80⁺ LCs (E) and CD86⁺ LCs (F) (dark gray filled, IMQ-untreated; black line, IMQ-treated; for CD80, n = 17, 4 independent experiments; for CD86, n = 13, 3 independent experiments). (G) Freshly isolated epidermal LCs were incubated at 37°C or 4°C (as control) with dextran-FITC for 45 minutes. Representative FACS analysis, histogram, and the percentages of dextran-FITC⁺ epidermal LCs (light gray line, IMQ-untreated at 4°C; dark graey filled, IMQ-untreated at 37°C; black line, IMQ-treated at 37°C; n = 12, 3 independent experiments). (H) Freshly isolated epidermal cells were in vitro cultured in the presence of Golgi Stop for 4 hours, and they were stained with anti–MHC-II, anti–CD45.2, anti–IL-23p19 Ab and analyzed by flow cytometry. Representative FACS analysis and percentages of IL-23⁺ LCs (n = 12, 3 independent experiments). Two-tailed Student’s t test was performed. The data are presented as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001.