Sp8 laser confocal microscope system

For evaluation of dermal lymphatic vessel contraction, mice were anaesthetized with isoflurane and hair on the dorsal side of the ears was removed using a sharp blade. The head and nose of the mouse was fixed to a customized head holder device and the left ear was glued to a customized plastic plate to prevent movement. TRITC-Dextran (1 µl of 10 mg/ml, 500 kDa, Sigma-Aldrich) was injected subcutaneously with a (30G) insulin syringe (BD Biosciences) or a Hamilton syringe. Mice were then immediately transferred to the imaging stage for time-lapse epifluorescence imaging using either the 20×/1.0 objective on a Leica SP8 laser confocal microscope system (Movie 1, images acquired every 1 s) or a Leica M205FA microscope with a PLANAPO 1.0× objective (Leica Microsystems) (Movies 2,3,4,5, images acquired with an interval of less than 1 s).

For immunofluorescence, embryos were fixed in 2% paraformaldehyde in PBS (pH 7.4) for 20 min and permeabilized using 0.5% triton X-100 in PBS for 20 min. The embryos were then incubated in blocking buffer (3% BSA in PBS) for 2 h followed by incubating with primary antibodies (diluted in blocking buffer) at 4°C overnight. After washing three times (15 min each) in washing buffer (0.5% tween-20, 0.5% triton X-100 in PBS), appropriate diluted fluorescent secondary antibodies were applied for 2 h. Embryos were then incubated in DAPI staining solution for 10 min. Apart from specially mentioned, all above procedures were performed at room temperature. The immunofluorescence staining was visualized by SP8 laser confocal microscope system (Leica, Wetzlar, Germany). Antibodies used are shown in Table 2. Nuclear was stained with DAPI staining solution (1:1,000, Beyotime, Shanghai, China, C1005). Quantification was performed by ImageJ.