G3282

To identify calcification, von kossa staining was performed according to the manufacturer’s instructions (G3282, Solarbio Life Science, China). Briefly, the cells were removed from the medium in a six-well plate, washed three times with saline solution, fixed for 30 min in 10% formalin, and rinsed three times with ddH₂O. The cells were then incubated with 5% silver nitrate solution at ambient temperature for 10 min under ultraviolet light until the color developed. The silver nitrate solution was discarded, and the cells were washed with ddH₂O for 3 min. Under light, the cells were treated with 3% sodium thiosulfate solution for 2 min and rinsed for 5 min. After hematoxylin staining for 5 min and treatment with 1% hydrochloric acid alcohol solution for 10 s, the slides were rinsed until they became blue. The dried coverslips were sealed with neutral gum. Finally, the slides were imaged under a microscope.

Von Kossa and ARS staining of aortic tissues were performed using the calcium staining kit (Von Kossa method, G3282, Solarbio Life Sciences, Beijing, China) and alizarin red S solution (G1450, Solarbio Life Sciences, Beijing, China). Aortic tissues were fixed in 10% neutral formalin prior to dehydration and embedding. Sections were embedded in 95% ethanol, placed vertically, and air-dried thoroughly. Von Kossa staining was performed according to the manufacturer’s instructions. For the ARS staining, the sections were then placed in a vat containing ARS solution and stained for 5–10 min followed by a quick rinse in distilled water. The sections were dehydrated in a conventional transparent manner and embedded in resin.

All embryos were fixed overnight in 4% paraformaldehyde, dehydrated through graded alcohols, embedded in paraffin, and sectioned at a thickness of 4 µm. For TRAP staining, sections were incubated with pure water at 37 °C for 2 h, placed in the prepared TRAP staining solution at 37 °C for dark staining for 1 h, and then counterstained with haematoxylin. For von Kossa staining, the sections were dropped with the prepared von Kossa silver staining solution and exposed to bright light for 15 min, followed by Hywave solution for 2 min, and finally counterstained with eosin. A TRAP kit (387A-1KT, Sigma, USA) and von Kossa kit (G3282, Solarbio, China) were used as described in our previous study^{9 (link)}. For immunohistochemistry, sections were subjected to antigen retrieval and then blocked with normal goat serum, followed by incubation overnight at 4 °C with the primary antibodies anti-CD14 (1:200, ab181470, Abcam), anti-F4/80 (1:200, ab300421, Abcam), anti-CSF1R (1:200, ab254357, Abcam), and anti-RANKL (1:200, ab45039, Abcam). Anti-CD31 (1:300, ab28364, Abcam) and goat anti-rabbit IgG H&L (1:200, ab150080, Abcam) were used.