The immobilized perilymphatic CCL21 gradient is diminished during CHS-induced skin inflammation. (A–E) Analysis of the immobilized perilymphatic CCL21 gradient stained in fresh (unfixed) CTR and CHS-inflamed ear skin (24 h after challenge). (A) Representative images showing the CCL21 and LYVE-1 signal at 20× magnification (scale bar: 50 μm). (B) Based on the LYVE-1 signal, a mask was generated to analyze the CCL21 intensity in relation to distance from the nearest LV. Scale bar: 100 μm. (C–E) Quantification of the CCL21 staining intensity as a function of the distance from the nearest LYVE-1⁺ LV. CCL21 staining intensity was measured at (D) 0 μm or (E) 30 μm from the LV. n = 6 mice per condition (4–6 images analyzed per mouse). The dotted horizontal line in D and E indicates the level of background (isotype) staining. (F–H) Analysis of intracellular CCL21 deposits, revealed by staining in PFA-fixed CTR and CHS-inflamed ear skin. (F) Representative images. Scale bar: 50 μm. Quantification of G the intracellular CCL21 staining intensity and (H) the number of CCL21 deposits present within lymphatics. Pooled data from n = 4 mice/condition with 3–6 images/ear skin are shown. Data from the same experiment (i.e., same mouse) in G and H are connected by a line and analyzed by paired Student’s t test.