Nbp1 43411

Immunostaining was performed as previously described (Wang et al., 2018 (link)). mLECs were identified by immunostaining with anti-LYVE-1 (NBP1-43411, Novus) and anti-VEGFR-3 (ab27278, Abcam) antibodies, as both of which are the selective markers for lymphatic endothelium. Briefly, cells were washed with PBS three times, fixed with 4% paraformaldehyde for 30 min, permeated with 0.1% Triton X-100 for 15 min, and then blocked with 5% bovine serum albumin. Cells or heart frozen sections were incubated with primary antibodies against LYVE-1 and VEGFR-3 (diluted 1:100) overnight at 4°C and then incubated with a red fluorescent secondary antibody (diluted 1:200; A0453, Beyotime) for 2 h at room temperature. The cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and are shown in blue. The images were analyzed using a Nikon microscope (Tokyo, Japan).

Heart tissues were fixed, dehydrated in 20% sucrose, and then embedded in optimal cutting temperature compound. Wheat germ agglutinin (WGA) staining was performed to estimate cardiomyocyte (CM) hypertrophy. For immunofluorescence, heart sections or cells were incubated with an anti-CD68 (ab125212, 1 : 200), anti-VEGFR-3 (ab273148, 1 : 200), anti-LYVE-1 (NOVUS, NBP1-43411, 1 : 200), or anti-VE-cadherin (ab205336, 1 : 200) antibody and then with fluorescently labeled antibodies (Life-Lab, 1 : 200, CN). DHE staining was performed to measure the reactive oxygen species (ROS) levels as previously described [23 (link)]. To quantify staining intensity, four sections from areas at least 60 mm apart in each heart were randomly selected. At least six nonoverlapping fields of each section were imaged with a fluorescence microscope.