Anti opn antibody

Formalin-fixed human biopsies were embedded in paraffin and cut into 3 mm sections. Sample slides were incubated with anti-OPN antibodies (Abcam, Shanghai, China) at a 1:75 dilution, followed by a 30 min incubation with an HRP-labelled polymer secondary antibody (Santa Cruz, Shanghai, China). Sections were viewed with a Nikon ECLIPSE E600 microscope (Nikon, Tokyo, Japan) using 10× objective lenses, and images were acquired with a SPOT INSIGHT™ digital colour camera, model 3.2.0 (Sterling Heights, Michigan, USA). Quantification of immunoreactivity was performed on digitally captured colour images saved as TIFF files and analysed using Image-Pro plus 6.0 (Media Cybernetics, Rockville, Maryland, USA). Blind immunohistochemistry analysis was conducted by a pathologist of Fudan University. These methods were performed in accordance with the approved guidelines from the Animal Ethics Committee of Fudan University.

RIPA lysis buffer (strong) RIPA (YEASEN, 20101ES60, USA) was used to extract proteins from mechanically stretched and unstretched AD‐VSMCs. The protein concentration was determined using the BCA method, and Western blotting was then used to detect protein levels. Briefly, protein extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were then blocked with 5% skim milk for 2 h and incubated with primary antibodies overnight at 4°C. After washing three times with TBST, the membranes were incubated with secondary antibodies for 1 h. Visualization was performed using an enhanced chemiluminescence detection system, and Photoshopcs3 software was used to analyze the relative band intensity. The primary antibodies used in this study were as follows: anti‐α‐SMA antibodies (Abcam, Cambridge, UK; cat. no. ab7817), anti‐SM22‐α antibodies (Abcam; cat. no. ab14106), anti‐OPN antibodies (Abcam; cat. no. ab8448), anti‐PCNA antibodies (Abcam; cat. no. ab29), anti‐KLF4 (Abcam; cat. no. 106629), anti‐MMP9 antibody (Abcam; cat. no. ab76003), anti‐GAPDH antibody (Abcam; cat. no. ab9485) and anti‐β‐actin antibody (Arigo; cat. no. arg62346).

Following sample harvest or micro-CT analysis, the fixed specimens were decalcified in 10% ethylenediaminetetraacetic acid solution (pH 7.4) at room temperature and processed for paraffin embedding. Paraffin blocks were sectioned at a thickness of 5 µm using a microtome. The calvarial sections were stained with hematoxylin and eosin (H&E) and Masson’s trichrome to examine the newly formed tissues. ImageJ was utilized to measure and analyze the new bone formed area in H&E stains and the red-stained area in Masson’s trichrome stains of the calvarial tissue sections. For immunohistochemistry, the calvarial sections were stained with anti-OCN (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and anti-OPN antibodies (Abcam, Cambridge, UK). Primary antibodies against anti-OCN (Cat# sc-365797, 1:200) and anti-OPN (Cat# ab63856, 1:200) were incubated at 4 °C overnight. The HRP-conjugated secondary antibodies (Vector Laboratories, Inc., Burlingame, CA, USA) against the primary antibody were applied for 1 h at room temperature, and the sections were incubated for 10 min with the diaminobenzidine substrate (ImmPACT® DAB; Vector Laboratories, Inc.) to detect signals. The OCN and OPN-positive area were evaluated by ImageJ (NIH) using the Colour Deconvolution 2 plugin.