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. 2016 Jul 27;19(4):513–524. doi: 10.1007/s10456-016-9523-8

Fig. 1.

Fig. 1

Lymphatic vessels on the pleural side of the diaphragmatic muscle develop and mature postnatally. Whole mounts of C57BL/6 mice show that on the pleural side of the diaphragmatic muscle, LYVE-1-positive vessel structures (green) co-stain for Prox-1 (red) at P7 (a–c) and for CD31 (red) at P5 (d–f). Wide-field images of LYVE-1 whole mounts demonstrate few LYVE-1-positive vessel structures at E16.5 and E18.5 and an expansion of the vessel network from P0 to P7 (g). $ mark unspecific staining of liver tissue. Dotted lines mark the area of the diaphragm that is used for whole-mount imaging and quantifications (g). Merged confocal images of lateral diaphragm segments stained for LYVE-1 show the expansion of the vessel plexus starting from P0 to 6 W (h). Stars mark the central tendon region. High-magnification confocal images allow the visualization of diaphragmatic lymphatic vessel sprouts at P7 (i arrows). Prox-1 (cyan), α-SMA (red) and CD31 (green) whole-mount stainings show valves (arrows) and SMC coverage of lymphatic vessels, located close to the thorax wall (j). CD31 (red) and LYVE-1 (cyan) diaphragm whole mounts of a 6-week-old Prox-1 GFP mouse show LYVE-1 down-regulation on diaphragmatic lymphatic vessels located close to the thorax wall (K-N). Scale bars a–f 100 µm, g and h 1 mm, i 50 µm, j 100 µm, k–n 50 µm. (Color figure online)