Specificity of novel antibodies (ALY) against mouse LYVE-1. (A) ALY2, 7, and 9 binding to LYVE-1-FLAG revealed by ELISA. Shown is absorbance at 570 nm. Each bar represents the mean ± SD of triplicate assays. (B) ALY7 binding to BaF/LYVE-1 cells was analyzed by FACS. The red line denotes binding of ALY7 to BaF/LYVE-1 cells. The black line denotes background staining seen when BaF/LYVE-1 cells were incubated with only secondary antibodies. (C-D) LECs in mouse embryos at E14.0 were analyzed by whole-mount immunohistochemical staining with anti-LYVE-1 mAb (ALY7). (E-F) The lymphatic endothelial network in E14.0 mouse embryos is visualized by VEGFR-3 expression. To detect VEGFR-3 expression, whole-mount X-gal staining of VEGFR-3^LacZ/+ mouse embryos was performed. Panels D and F show higher magnification views of the black square in the upper limb region of panels C and E, respectively. (G) Section of lymphatic vessel in the skin tissues of the VEGFR-3^LacZ/+ embryo. X-Gal staining shows that VEGFR-3⁺ (blue color) are identical to LYVE-1⁺ (brown color). (H-L) Section of the E14.0 mouse vascular system in body wall was stained with ALY7 (red color in panels H and J), anti-mouse podoplanin mAb (green color in panels I and J), isotype-matched rat IgG (red color in panel K), and isotype-matched hamster IgG (green color in panel L). LYVE-1⁺ lymphatic vessel (LV in panel J) expresses podoplanin, but blood vessel (BV in panel J) does not. Isotype-matched control antibodies showed no staining in any sections (K-L). Scale bars indicate 10 μm (G) and 20 μm (H-L), respectively.