Vwf antibody

Aorta patches were prepared as described in the section Aorta sample preparation. Next, the aorta patches were fixed with 1.5% formaldehyde for 10 min. The samples were then washed 3 times in PBS and incubated with a solution of blocking peptide for 30 min. The samples were then gently washed in PBS and incubated with vWF Antibody (Santa Cruz Biotechnology sc-53466, 1:50) for 1 h at RT. Afterwards, the ECs were gently rinsed with PBS and incubated with m-IgGκ BP-CFL 594 (Santa Cruz Biotechnology). Additionally, cell nuclei were stained by Hoechst (ThermoFisher). Images were acquired with a Zeiss LSM710 confocal microscope with 40× 1.4 NA PlanApo objective lens and using the Zen 2012 Black Edition software supplied by the manufacturer.

Lungs were inflated, harvested, fixed in 2% paraformaldehyde, and embedded in paraffin. To visualize the pulmonary medial arterial wall of pulmonary vessels, lung sections were immunostained with a polyclonal rabbit anti-α-smooth muscle actin (α-SMA) antibody at a dilution of 1:200 (Abcam, USA) for 3 h at 4°C. To quantify the density of pulmonary vessels, a polyclonal rabbit anti-von Willebrand factor (vWF) antibody (Santa Cruz Biotechnology, USA) was employed as an endothelial marker at a dilution of 1:400. The slides were washed and incubated with biotinylated goat anti-rabbit IgG for 1 h and washed again. After washing in PBS, the signal was detected with 3,3'-diaminobenzidine (Dingguo, China). α-SMA-stained tissue sections were captured at ×400 magnification and the thickness of the vessel wall was calculated using the following equation: ((area1−area2)/area1)×100, where area1 and area2 are the areas within the external and internal boundaries of the α-SMA layer, respectively. To quantify vessel density, the number of vWF-positive vessels per high-power field was counted at a magnification of ×100.