Ab33168

Deparaffinizing, antigen retrieval, and blocking of non-specific background were performed as described previously 22 (link) and sections were incubated with primary antibodies overnight at 4°C. After washing three times with PBS, and incubated with fluorescence-labeled secondary antibodies for 1 hour at room temperature in the dark. Slices were washed three times in PBS, and then counterstained with DAPI (Sigma-Aldrich, St Louis, MO, USA). The primary antibodies were as follows: anti-sarcomeric alpha actinin (1:200; ab9465, Abcam, UK), anti-phosphorylated Histone 3 (1:1000; 3377, Millipore, USA), anti-Ki67 (1:200; ab16667, Abcam, UK), anti-Aurkb (1:100; A5102, Sigma, USA), anti-CDH5 (1:100; ab33168, Abcam, UK) and anti-Vimentin (1:100; ab92547, Abcam, UK). The secondary antibodies were from Invitrogen: anti-rabbit Alexa Fluor 488-conjugated (1:400; A21206, Invitrogen, USA), anti-mouse Alexa Fluor 594-conjugated (1:400; A21203, Invitrogen, USA). Fluorescence was observed under a ZEISS LSM800 confocal laser scanning microscope (Carl Zeiss, Inc., Jena, Germany). We used High Content Screening System (Opera Phenix) to count cell proliferation ratio.

For heart tissue staining, frozen myocardial tissue was horizontally sliced into 5 μm section and mounted on glass slide. Both sections and cells were fixed in 4% paraformaldehyde for 15 min, permeabilized with Triton X-100 (0.1%) and blocked with solution containing 5% bovine serum before applying primary antibody. Specimens were incubated with anti-α-smooth muscle actin (α-SMA) (Abcam, catalog #ab5694; 1:100), anti-fibroblast- specific factor 1 (FSP1) (Abcam, catalog #S100A4; 1:200), anti-VE-cadherin (Abcam, catalog #ab33168; 1:300), anti-CD31(Abcam, catalog #ab24590; 1:200) and anti-β-catenin (Abcam, catalog #ab32572; 1:200) overnight at 4 °C, washed three times in PBS and incubated with the secondary antibody (Life Technologies, catalog #A21207; 1:200) under light-protected conditions for one hour at room temperature and counterstained using 4,6-diamino-2-phenyl indole (DAPI) (KeyGen Biotech, catalog #KGA215–10). Cardiovascular endothelial cells were identified by staining with the lectin BS1-B4 (Sigma, catalog #L-2895) under light-protected conditions at room temperature for 3 hours at and the cover slips were mounted on slides using 50% glycerin. Then, the sections and cells were observed under a fluorescence microscope (Olympus, Tokyo, Japan) or confocal laser scanning microscope (Olympus, Tokyo, Japan).

Cells cultured in the FEPM devices were fixed with 4% paraformaldehyde (PFA, Nacalai Tesque, Kyoto, Japan) for 20 min, washed with PBS, and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, USA) for 15 min. The cells were then incubated with blocking buffer containing 1% bovine serum albumin (BSA, Sigma-Aldrich) for 30 min at room temperature and incubated with antibodies directed against vascular endothelial (VE)-cadherin (Abcam, ab33168, dilution 1:200, Cambridge, UK) or Claudin (Abcam, ab15098, dilution 1:200) overnight at 4 °C, followed by PBS washes. Subsequently, Alexa 568-conjugated secondary antibody (Abcam, ab175471, dilution 1:500) was introduced into the channels and incubated for 1 h at room temperature. The cells were co-stained with 4’,6-diamidino-2-phenylindole (DAPI, Invitrogen, D1306, Waltham, USA). Cross-sectional images were obtained at 2 µm intervals in the vertical direction using a confocal microscopy (FluoView FV1000 confocal, Olympus, Tokyo, Japan) with the 10× objective.

Total proteins were extracted from zebrafish larvae or cell lysates with a RIPA lysis kit (C1053; Applygen Technologies Inc., Beijing, China), separated using 12% SDS-PAGE, and transferred to a nitrocellulose membranes (T41524; PALL, Mexico), which were blocked with 5% milk in Tris-buffered saline for 1 h at room temperature and then incubated with primary antibodies anti-LOX-1 (ab60178, Abcam), anti-VE-cadherin (ab33168, Abcam), anti-N-cadherin (13A9, mAb #14215, CST), anti-α-SMA (ab7817, Abcam), anti-LC3B (M186-3, MBL), anti-p62 (PM045, MBL), anti-LAMP2 (sc-18822, Santa Cruz), or anti-β-actin (HC201-01, ZSGB-BIO Co) overnight at 4 ºC. After washing, the blots were incubated with goat anti-mouse or goat anti-rabbit immunoglobulin G (ZSGB-BIO) for 1 h and protein bands were visualized by an immobilon western chemiluminescent horseradish peroxidase substrate (Millipore, Billerica). The western blot images are representatives of 4-6 independent experiments with corresponding histograms.

The cells were tiled on coverslips, incubated at 37 °C overnight, permeabilized with 0.1% Triton X-100 and blocked with 5% FBS. Then, the cells were incubated with primary antibodies against S1PR1 (ab11424; Abcam; 1:50), VE-cadherin (ab33168; Abcam; 1:50), and β-catenin (ab32572; Abcam; 1:100). After incubation with fluorophore-conjugated secondary antibodies, the nuclei were counterstained with 4',6-diamidino-2-phenylindole(DAPI) (Sigma). Images were acquired by fluorescence microscopy (Nikon, Japan).