Diaminobenzidine substrate chromogen system

After organ culture for 7 days, thoracic aorta segments were washed with PBS and fixed with 10% neutral buffer formalin. The specimens were then embedded in paraffin, and 5μm cross-sections were cut. The sections were deparaffinized and H&E staining was performed. Arterial medial calcification was visualized using Alizarin Red S staining as previously described ^{23 (link)}. For immunohistochemistry, the sections were deparaffinized, followed by treatment with citrate buffer for antigen retrieval and 3% H₂O₂. The sections were blocked with Dako serum-free blocking solution (Dako North America, USA) and incubated with primary antibody for overnight at 4°C. The primary antibodies included SM22α (Abcam, ab14106), Osterix (Abcam, ab94744). Subsequently, the sections were incubated with biotinylated secondary antibodies for 30 min at room temperature. Avidin-biotinylated enzyme complex (Vector Laboratories, USA) and a diaminobenzidine substrate chromogen system (Dako North America, USA) were used for detection. Sections were counterstained with hematoxylin. The images were captured by Olympus DP260.