Validation of the differential expression of selected cell surface proteins in CHS-inflamed LECs. Differential expression of selected cell surface molecules was validated at the protein level by FACS analysis and by immunofluorescence. (A) FACS analysis was performed on single-cell suspensions obtained from enzymatically digested ear tissue. (Upper panels) The staining and gating scheme. (Lower 2 rows) Histograms of candidate gene expression in LECs (CD45⁻CD31⁺podoplanin⁺ cells). Black line indicates candidate gene; and gray, tinted line, isotype control. Representative plots from 3 or 4 different experiments are shown. The number in the top right of each image represents the ΔMFI, defined as the difference in the median fluorescent intensity between the candidate and the corresponding isotope control staining. (B) Summary of the ΔMFI values measured in all experiments. Data points (control [CTR] − inflamed [INFL]) from the same experiment are connected by a line. (C) Immunofluorescence was performed on cryosections, obtained from uninflamed (control) or CHS-inflamed ears 48 hours after challenge. Sections were stained for candidate genes (red) and costained with LYVE-1 (green) to outline lymphatic vessels. uPAR (red) was found to be up-regulated in CHS-inflamed ear skin (↑), whereas NrCAM (red) was down-regulated (↓). Representative images from 2 different experiments are shown. Scale bar represents 50 μm.