Kga215 10

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).

Mice were intracardially perfused with cold saline followed by 4% paraformaldehyde (PFA, in 0.1 M phosphate-buffered saline (PBS); pH 7.4) and then brains were collected and postfixed for 6 h in 4% PFA at 4 °C. Brains were sectioned with a vibratome or freezing microtome at 30 μm in the coronal plane and blocked in 0.1 M PBS containing 10% serum and 0.3% Triton X-100 (Sangon Biotech) for 2 h at room temperature. Sections were treated with 10 μg/ml proteinase K (VICMED, diluted in Tris HCl buffer containing 10 mM Tris-HCl and 100 mM NaCl, pH 7.8). Sections were then incubated with anti-pSer129 α-synuclein (1:2000, diluted in 0.1 M PBS containing 1% serum and 0.1% Triton X-100) overnight at 4 °C, followed by incubation with appropriate secondary antibodies for 1 h and 4,6-diamidino-2-phenylindole (DAPI; nuclear stain; KeyGEN BioTECH, KGA215-10) for 10 min at room temperature. Labeled sections were imaged on a confocal scanning microscope (Zeiss, LSM710). The density of pSer129 α-synuclein-positive signals in different layers was analyzed using the ZEN Blue 3.0 software.