(A) Lateral back skin of BALB/c mice was exposed to AFL, dissected right after treatment, and subjected to histological analysis. Representative skin MC (arrow) and thermal damage zone are shown. Edges of skin MC and thermal damage zone were marked with dashed blue lines. Scale bar: 100 μm. (B–D) Lateral back skin of BALB/c mice was exposed to AFL, intradermally injected with Alum adjuvant, or left nontreated. Skin was dissected 24 hours later. Total proteins were extracted, pooled (n = 3), and subjected to membrane immunoblotting analysis of cytokine levels. Representative immunoblotting images (B). The commonly and differentially stimulated cytokines by AFL and ID Alum adjuvant (C). Mean pixel intensities of different cytokines in B were measured by ImageJ (NIH) and compared (D). The duplicate cytokine spots within the rectangles in B are labeled with numbers to indicate increased cytokine levels after treatment. The corresponding cytokine name can be found in D (x axis). (E) Innate immune cell recruitment after AFL treatment and ID Alum adjuvant injection. Lateral back skin of mice was exposed to AFL or intradermally injected with Alum adjuvant. Skin was excised at different time points. Single-cell suspensions were prepared followed by immunostaining and flow cytometry analysis. Percentage of neutrophils, monocytes, macrophages, eosinophils, and mDCs in total skin cells is shown. Gating strategies are shown in Supplemental Figure 2E. n = 4. Two-way ANOVA with Bonferroni’s posttest was used to compare differences between groups at different time points (E). *, P < 0.05; **, P < 0.01; ***, P < 0.001.