IL-7 and its receptor chains are expressed in LECs in vivo. (A-B) FACS sorting was used to isolate LECs and BECs from single-cell suspensions generated from enzymatically digested mouse ear skin. qPCR was performed on the extracted, reversely transcribed and amplified complementary DNA material. (A) Depiction of the gating scheme used to isolate BECs (CD45⁻CD31⁺podoplanin⁻) and LECs (CD45⁻CD31⁺podoplanin⁺). (B) qPCR analysis revealed that IL-7 and its receptor chains were more highly expressed in LECs compared with BECs. Pooled data from 3 experiments are shown. (C-E) To validate the expression of candidate genes in LECs (gated on CD45⁻CD31⁺podoplanin⁺ cells) and BECs (gated on CD45⁻CD31⁺podoplanin⁻ cells) at the protein level, FACS analysis was performed on ear and brachial LN single-cell suspensions. (C) CD132 was preferentially expressed in LECs compared with BECs, whereas (D) IL-7Rα was similarly expressed in LECs and BECs. (E) No expression of IL-7Rα was detected when performing FACS analysis on ear skin and LN single-cell suspensions from IL-7Rα^−/− mice. Representative plots from 3 different experiments are shown. (F) FACS analysis performed on single-cell suspensions generated from the ears of IL-7-CrexR26-EYFP mice confirmed YFP expression in most LECs (gated on CD45⁻CD31⁺podoplanin⁺), but not in BECs (gated on CD45⁻CD31⁺podoplanin⁻). Representative FACS plots showing YFP expression in LECs and BECs are shown. (G) A summary of the percentages of YFP-expressing LECs and BECs from all experiments (n = 3) is shown. (H-I) Confocal laser scanning microscopic analysis of ear skin whole mounts prepared from IL-7-CrexR26-EYFP mice detected YFP expression in virtually all LYVE-1⁺ LVs (H), but not in MECA-32⁺ blood vessels (I). Scale bar represents 100 μm. **P < .01.