S1P promoted in vivo lymphangiogenesis. Ten days after subcutaneous injection of Matrigel containing none, VEGF-C (1 μg), or S1P (0.4 μg), in C57BL/6 mice (5 mice per group), the Matrigel was removed, fixed, embedded in paraffin, sectioned at 4 μm, and immunostained. (A) Cross-sections of Matrigel were stained by H&E. Scale bars represent 50 μm. (B,C) Lymphatic vessels in Matrigel were immunostained for podoplanin (red). Representative photographs and the number of podoplanin-positive lymphatic vessels per field are shown in panels B and C, respectively. Scale bars represent 200 μm. (D) Lymphatic vessels in Matrigel were immunostained for Prox-1 (green) and podoplanin (red). Arrows show sprouting lymphatic vessels. Scale bars represent 50 μm. (E,F) Proliferating lymphatic endothelial cells in Matrigel were double immunostained for PH3 (green) and LYVE-1 (red). Representative photographs are shown in panel E. Scale bars represent 20 μm. The number of LYVE-1⁺ (□) or LYVE-1⁺/PH3⁺ (■) vessels per field were counted (F). (G) Uptake of injected FITC-dextran (2000 kDa) into newly formed lymphatic vessels was visualized using confocal microscope. Scale bars represent 100 μm. All values are expressed as means (± SEM). ** indicates statistically significant difference (P < .01).