G1105

Femoral-head samples were fixed for 48 h in 4% paraformaldehyde and decalcified for 4 weeks in 10% ethylenediaminetetraacetic acid (G1105, EDTA, Servicebio, China). Then, the samples were embedded in paraffin and cut into 6 μm thick slices. H&E staining was performed to observe the general view of specimens and to evaluate the trabecular structure. For TRAP staining, paraffin sections were stained with a TRAP staining kit (G1050, Servicebio, China). Briefly, the paraffin sections were stained with tartrate buffer containing naphthol AS-BI phosphate and pararosaniline chloride at 37 °C for 1 h in darkness and counterstained with hematoxylin. Section images were acquired using an IX71-SIF microscope (Olympus, Japan). IHC staining was performed to define the expression of CST, as well as osteogenesis-, vascular-, and pathway-related markers. In brief, sections were dewaxed and gradient hydrated to retrieve antigens. Then, primary antibodies, including CST, osteocalcin (ab133612), VEGFA (ab52917), and CD31 (ab9498), and corresponding secondary antibodies (all from Abcam Cambridge, UK) were incubated. The chromogenic reaction was induced by a DAB Kit (Beyotime, China). The tissue sections were observed with an IX71-SIF microscope (Olympus, Japan). IHC-positive cells and areas were measured using ImageJ and counted by two independent observers.

Mice’s liver and tibia were fixed in 10% neutral-buffered formalin solution. Tibia was decalcified in EDTA decalcified fluid (G1105, Servicebio), followed by paraffin embedding. Then, the livers and tibia were cut into 5 μm-thick sections and stained with hematoxylin and eosin (HE). The degree of lipid infiltration on liver HE staining was scored on a scale of 0–4 with 0 being normal healthy tissue and 4 being the worst, as described previously [17 (link)]. Marrow adipocyte area and number quantification were analyzed according to Styner and colleagues [18 (link)].