Anti α smooth muscle actin α sma

Left upper lung tissues were fixed with 4% paraformaldehyde, paraffin-embedded, and sliced with 4 µm thickness. Lung tissue sections were stained with hematoxylin and eosin (H&E). To determine the extent of collagen deposition in pulmonary arteries, Sirius red staining and Masson trichrome staining was performed and quantified by modified ashcroft scoring system [15 (link)16 (link)]. For immunohistochemisty, the tissue sections were blocked with peroxidase blocking agent for 5 min, washed with Tris-HCl with Tween (TBST), and blocked with protein blocking serum free buffer for 5 min. Then, sections were incubated with anti-α-smooth muscle actin (α-SMA) (Sigma-Aldrich Co.) or anti-von Willebrand Factor (vWF) (Millipore, Temecula, CA) overnight at 4℃ followed by washing with TBST for 5 min. Enough labelled polymer conjugated with secondary antibodies (Dako, Carpinteria, CA) were applied to the slides for 30 min, followed by washing with TBST for 5 min. Peroxidase activity was detected with the ready-to-use AEC+substrate chromogen (Dako). At least twenty arteries of 15-100 µm per each rat were evaluated in α-SMA stained slides through a light microscope (imager M1, Carl Zeiss, Jena, Germany) at ×400 magnification and quantified by image J software.
The medial wall thickness is calculated as follows:
Wall thickness=(total area of artery–lumen area of artery)/total area of artery

As described in detail previously [20 (link)], samples were fixed in 4% paraformaldehyde and blocked with normal goat serum. For smooth muscle and the endothelial content staining, anti-α smooth muscle actin (αSMA) (1:400; Sigma Aldrich Co.), anti-endothelial nitric oxide synthase (eNOS) (1:100; Becton, Dickinson and Company, Franklin Lakes, NJ, USA), and anti-von Willebrand factor (vWF) (1:400; Abcam, Cambridge, UK) were used. For neuronal staining, anti-neuronal nitricoxide synthase (nNOS) (1:100; Novus Biologicals, Centennial, CO, USA) was used. The nucleus was stained using 4,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA). Goat anti-mouse and goat anti-rabbit (1:400; Thermo Fisher Scientific) were used as secondary antibodies.