G8550

Calcification of mouse
VSMCs and aortas was visualized by Alizarin Red S staining. The cells
were washed three times with PBS and fixed with 4% paraformaldehyde
for 10 min after incubation. Next, cells were washed three times with
PBS and then stained by 2% Alizarin Red S (pH 4.2, G8550, Solarbio,
Beijing, China) for 5 min. Finally, the cells were washed with deionized
water to remove uncombined dye and then imaged using an optical microscope
(DM4000B, Leica, Germany) with Leica Application Suite X software
version 3.7. To visualize calcification of aortic tissues, the aortas
were bluntly isolated to remove the external connective tissue and
then fixed with 4% paraformaldehyde for 24 h. Besides, the aortas
were incubated overnight with 0.004% (m/v) Alizarin Red S dissolved
in 1% KOH in dark at room temperature and then washed with 2% (m/v)
KOH for 5 min.^{50 (link)} Positively stained samples
displayed a red color. Statistical analysis was performed via ImageJ
Pro Plus 6.0.

The hBMSCs from patients with femoral head necrosis secondary to senile femoral neck fractures were selected as the experimental subjects. Cells were seeded on 6-well plates. When cells reached 60–70% confluence, the medium was changed with the osteogenic differentiation medium to induce differentiation. The osteogenic differentiation medium was freshly prepared before use, which contained basic medium, 10% fetal bovine serum, 10 nmol/L dexamethasone (D8040; Beijing Solarbio Co., Ltd., China), 10 nmol/L ascorbic acid (A8100; Beijing Solarbio Co., Ltd., China), 50 mol/mL of glycerol phosphatide (Sigma-Aldrich, USA), 1% penicillin–streptomycin and 1% HEPES (H8090; Beijing Solarbio Co., Ltd., China). After 21 days of osteogenic differentiation, hBMSCs in the 6-well plate were washed with PBS and fixed with neutral formalin for 30 min. The fixation fluid was discarded, and the cells were rewashed with PBS. One milliliter of ARS agent (G8550; Beijing Solarbio Co., Ltd., China) was added to the wells for staining for 5 min. Then cells were washed and dried. The staining effect was observed under a microscope (DYF-880; Shanghai Dianying Optical Instrument Co., Ltd., China).