Oil red o staining solution

Oil Red O staining was performed as previously described (Ma et al., 2020 ). In brief, a Leica CM3050S cryostat was used to perform 10-μm-thick cryosections of monkey skeletal muscle embedded in Tissue-Tek O.C.T compound. After air-drying, the sections were fixed in a solution containing 4% PFA at room temperature (RT) for 10 min, and then stained in a freshly prepared Oil Red O staining solution (Sigma-Aldrich) for 30 min, rinsed with tap water and counterstained with hematoxylin. Images were taken with PerkinElmer Vectra Polaris. Image J was used to quantify the percentage of Oil Red O-positive area.

Oil Red O staining was performed to determine the content of lipid droplets in differentiating 3T3-L1 cells. The adipocyte differentiation program as described previously was established, and cells were exposed to each tested compound at 5 µM for 48 h after obtaining 100% confluence. Cells exposed to 0.5% (v/v) DMSO and 20 µM of oxyresveratrol served as untreated vehicle and positive controls, respectively. Tested compounds were diluted in the differentiation medium before testing, in which the concentrations of DMSO were restricted to be lower than 0.5% (v/v). After the treatments, differentiating 3T3-L1 cells on day 8 of the differentiation program were fixed with 10% formalin and incubated with the Oil Red O staining solution (Sigma Aldrich, St. Louis, MO, USA) at an ambient temperature for 1 h. Cells were then washed thrice with distilled water and 60% (v/v) isopropanol (Sigma Aldrich, St. Louis, MO, USA) to remove the excess dye. Oil Red O-stained 3T3-L1 cells were observed using a light microscope (Nikon Ts2, Tokyo, Japan). The cellular Oil Red O content was extracted using absolute isopropanol and measured for absorbance at a 510 nm wavelength by the microplate reader. The percentage of Oil Red O-stained cells was calculated after normalization with the total cellular protein content determined by a BCA protein assay kit (Thermo Scientific, Rockford, CA, USA).

The ADSCs were seeded into a 6-well plate at a density of 5 × 10⁴ cells/ml. When the cells reached approximately 80% confluence, the medium was replaced by either adipogenic inducer medium or osteogenic inducer medium. The adipogenic inducer medium consisted of DME/F12 containing 1×10⁻⁶ mol/L dexamethasone (Sigma, USA), 10 mg/L insulin (Sigma, USA), 0.5 mmol/L 3-isobutyl-1-methylxanthine (Solarbio, Shanghai, China), and 0.2 mmol/L indomethacin (Solarbio, Shanghai, China). The osteogenic inducer medium is DMEM/F12 containing 1 × 10⁻⁶ mol/L dexamethasone (Sigma, USA), 50 μg/mL ascorbic acid (Solarbio, Shanghai, China), and 10 mmol/L β-sodium glycerophosphate (Solarbio, Shanghai, China). The inducer medium was changed every 3 d. After 15 d of induction, the cells were tested for the conversion of adipocytes with oil red O staining solution (Sigma, USA). After 21 d of induction, cells were stained with alizarin red staining (Ybscience, Shanghai, China) solution to detect the conversion to bone cells [29 (link)].

Mineral deposition was evaluated by von Kossa and Alizarin Red staining. For von Kossa staining, formalin-fixed cells were incubated with 5% AgNO₃ for 15 minutes followed by 5 minutes of 1% pyrogallol. After fixation with 5% sodium thiosulfate solution, cells were rinsed with H₂O and images were taken. For Alizarin Red staining, formalin-fixed cells were incubated with 2% Alizarin Red for 1 h (#A5533, Sigma), rinsed with H₂O and images were captured. Proteoglycan synthesis was evaluated using Alcian Blue staining. Cells were fixed in 4% PFA and stained with 0.1% Alcian Blue in 0.1 N HCl overnight. Cells were washed and stored in 70% EtOH for image capturing. Alcian Blue was extracted using 6 M guanine-HCl and the OD was measured at 630 nm. Lipid droplet formation was assessed by Oil Red O staining. Formalin-fixed cells were rinsed in 60% isopropanol for 2 minutes. Subsequently, cells were incubated in Oil Red O staining solution (Sigma) prepared following the instruction of the manufacturer for 30 minutes, then rinsed with H₂O before images were taken. Oil Red O was extracted by addition of isopropanol containing 4% NP40 and the OD was measured at 540 nm.

Briefly, after the treatment protocol, 5 × 10⁴ cells were washed with PBS three times and incubated with methanol for 15 min at room temperature, and then fixed in paraformaldehyde solution (PFA, 4%) for 20 min. After washing, cells were kept in Oil Red O staining solution (Sigma-Aldrich) (0.1%) for 30 min. Finally, after washing, images were captured by light microscopy (IM-3/ OPTIKA Italy) equipped with a CCD camera (TrueChrome II).