Formaldehyde solution

BMSCs were seeded into 24-well plates and cultured with osteogenic differentiation medium (Cyagen Biosciences, USA) after resveratrol treatment. After 3 days or 7 days cell culture, cells were fixed with 4% formaldehyde solution (Boster Biological Technology co. ltd, China) for 30 min and washed twice with PBS. Then cells were stained with alizarin red dye (Cyagen Biosciences, USA) for 5 min, washed twice with PBS, and imaged in the light field to observe the calcium nodules. To quantitatively analyze the calcification, 2 mL cetylpyridinium chloride (Sigma-Aldrich, USA) was added into each well to dissolve the calcium nodules. The absorbance at 562 nm was measured.

Lung samples of mice under different treatments were harvested at indicated time points, fixed in a 4% formaldehyde solution (Boster, Wuhan, China), and subsequently embedded in paraffin. The lung samples were then cut into 6 μm-thick sections, followed by H&E and Masson’s trichrome staining. The images of lung sections were taken by a Panoramic MIDI slide scanner (3DHISTECH Kft., Budapest, Hungary). The collagen depositions of the lung were evaluated by quantifying the ratio of blue area in the Masson’s trichrome section using Image J software.