Anti osteocalcin

Cells were digested using trypsin and collected. Cells were incubated with extraction lysis buffer containing 50 mM Tris-HCl 8.0, 150 mM NaCl, 0.5 mM EDTA, 0.25% Nonidet P(NP)-40 and incubated for 30 min at 4°C. After centrifugation at 12000g, 4°C for 10min, supernatant was collected and fractionated by 10% SDS-PAGE gel. After electrophoresis, blots were transferred to PVDF membrane (Life Technologies, Grand Island, NY, USA). The membrane was blocked with PBS containing 5% BSA (Sigma–Aldrich, St. Louis, MO, USA) for 1 h and incubated with the following primary antibodies: anti-osteocalcin (1:2000; Abcam), anti-Collagen I (1:1000; Abcam), anti-RUNX2 (1:1000; Abcam), anti-CTGF (1:1000; Abcam), anti-total Smad1/5/8 (T-Smad1/5/8; 1:1000; Abcam), anti-phospho Smad1/5/8 (p-Smad1/5/8; 1:1000; Abcam) or anti-β actin (1:4000; Abcam) at 4°C overnight. After three washes with PBS, membrane was incubated with horseradish-peroxidase (HRP) conjugated donkey anti-rabbit (1:10000; Abcam) for 2 h at room temperature. The membrane was incubated with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA), and exposed to film.

Immunohistochemical analyses of aorta were performed using paraffin sections. The primary antibodies, anti-osteopontin (1:100, Immuno-Biological Laboratories), anti-osteocalcin (1:100, Abcam) and anti phospho-Smad1 (1:200, Cell Signaling) were incubated overnight at 4°C. Revelation was performed with Universal vectastain ABC kit and ImmPACT AEC (Vector Laboratories) following the instructions of suppliers. Then, the sections were mounted in an aqueous mounting medium, VectaMount ^™ AQ (Vector Laboratories) and examined under a light microscope (Nikon Eclipse TE300). Quantification was carried out as for histological staining.

Cell samples were fixed in 4% (v/v) paraformaldehyde solution (MultiSciences Biotech, Hangzhou, China) at room temperature for 15 min. Endogenous peroxidase was inhibited by immersion in a 0.3% (v/v) hydrogen peroxide (H₂O₂) in methanol bath for 15 min. After washing, nonspecific binding was blocked with 5% (m/v) bovine serum albumin in PBS. The cells were incubated with diluted primary antibody Anti-SSEA4 antibody (15 μg/ml; Abcam), Anti-TRA-1-60 (R) antibody (1 : 100; Abcam), Anti-OCT4 antibody (5 μg/ml; Abcam), Anti-NANOG antibody (1 : 1000; Abcam), Anti-SOX2 antibody (1 μg/ml; Abcam), Anti-FABP4(1 : 1000; Abcam), Anti-PPAR gamma (5 μg/ml; Abcam), Anti-RUNX2 (1 : 500; Abcam), Anti-Osteocalcin (10 μg/ml; Abcam), Anti-human AFP (1 : 200; Abcam), Anti-human CK18 (2 mg/ml, 1 : 400; Abcam), Anti-human CK19 (1 : 400; Abcam). According to the manufacturer's instructions, detection of primary antibody was performed using horseradish peroxidase-conjugated secondary antibodies (1 mg/ml, 1 : 1000; Abcam). Peroxidase activity was revealed by a 3- to 5-min exposure to diaminobenzidine tetrahydrochloride solution (DAB kit, Vector Labs, Burlingame, CA, USA). The fixed cells were then washed, counterstained with hematoxylin for 1 min, mounted and observed under an inverted phase-contrast microscope (ECLIPAS TS100, Nikon, Tokyo, Japan).

Histological sections were treated with xylene, rehydrated through a graded ethanol series, and pre-treated in citrate buffer (pH = 6.0) 10 min at 98°C. The primary antibodies: cleaved caspase-3 (9664, Cell Signaling, USA), cleaved caspase-6 (9761, Cell Signaling, USA), cleaved caspase-7 (9491S, Cell Signaling, USA), cleaved caspase-8 (8592, Cell Signaling, USA), cleaved caspase-9 (9509, Cell Signaling, USA), and osteocalcin (ab93876, Abcam, UK), were diluted (anti-caspase-3: 1:50, anti-caspase-6: 1:50, anti-caspase-7: 1:50, anti-caspase-8: 1:200, anti-caspase-9: 1:50, anti-osteocalcin 1:100) and applied overnight at 4°C. Alexa Fluor® 488 (A11034, Thermo Fischer, USA) or Alexa Fluor® 594 (A11037, Thermo Fischer, USA) were diluted 1:200 and then applied for 40 min at room temperature (RT). Nuclei were visualised by ProLong Gold Antifade reagent with DAPI (Thermo Fischer, USA).