Masson staining

Liver tissues from the rodents were fixed in 10% formalin for 48 h, dehydrated via an ethanol gradient, and, then, embedded in paraffin wax. The paraffin-embedded tissues were sliced into 4 μm sections, with a microtome (Leica, Wetzlar, Germany). In accordance with the instructions of the manufacturer, liver steatosis and liver fibrosis were measured by hematoxylin and eosin (H&E) as well as Masson staining (Solarbio, Beijing, China). The frozen-compound-embedded tissues were sliced into 10 μm sections and used for Oil red O staining (Sigma), to reveal the presence of fat droplets in the liver. The sections were epimerized with 0.3% Triton X (Solarbio) for 3 min, rinsed with phosphate-buffered saline (PBS), and, then, stained with the Oil red O solution for 45 min at 65 °C. The samples were washed twice with PBS and, then, fixed with 10% formalin for 15 min, to observe the cells. Oil red O staining was performed, as described previously. All slides were mounted with resin or a glycerol-gelatin mix and examined under an optical microscope (Olympus, Tokyo, Japan).

Calvaria bone samples were decalcified in 0.25 M EDTA (pH 7.4) for 10 days and then immersed in PBS for 2 h. After dehydration in gradient alcohol (70, 80, 90, 95, and 100%), samples were embedded in paraffin and sectioned at the thickness of 5 μm. Sections were stain with hematoxylin and eosin (H&E), Masson staining (Solarbio, China) and imaged using a lighting microscope (BX43, OLYMPUS, Japan). Masson sections (n = 5 for each group) were analyzed for the total tissue area and mature bone area of the defects. The kidney and liver of the mice were also harvested for H&E staining as the procedure above.