The immobilized perilymphatic CCL21 gradient is diminished during TPA-induced skin inflammation. The skin of one ear of each mouse was inflamed by topical application of the irritant TPA. Experiments with both ears, i.e., the uninflamed (CTR and the TPA-inflamed ear, were performed 1 day later. (A–D) Analysis of the extracellular, immobilized perilymphatic CCL21 gradient stained in fresh (unfixed) CTR and TPA-inflamed ear skin. (A) Representative images showing the CCL21 and LYVE-1 signal at 20× magnification (scale bar: 50 μm). (B) Quantification of the CCL21 staining intensity as a function of the distance from the nearest LYVE-1⁺ LV. CCL21 staining intensity was measured at (C) 0 μm or (D) 30 μm from the LV. n = 3 mice per condition (4–6 images analyzed per mouse). The dotted horizontal line in C and D indicates the level of background (isotype) staining. (E) Elution assay: CTR and TPA-inflamed ear skin were placed in medium overnight, and the amount of CCL21 protein eluted into the medium was determined by ELISA. (F and G) Quantification of (F) plasminogen and (G) plasmin activity in tissue protein extracts generated from CTR or TPA-inflamed ear skin. n = 6–7 mice per condition. All graphs: unpaired Student’s t test.