Alkaline phosphatase

Sarpogrelate was obtained from Shanghai Linebon Ltd., Shanghai, China. Bromocriptine was sourced from Novartis, Italy. Cabergoline was purchased from Pfizer, Italy. Domperidone was purchased from Jamjoom Pharmaceuticals Co., KSA. Alloxan was obtained from Sigma-Aldrich (St Louis, MO, USA). Urea, creatinine, alkaline phosphatase, aspartate aminotransferase activity, LDH-1 assay kits, and the TNF alpha ELISA kit were obtained from Abcam, USA. The troponin I test kit was obtained from Encode Medical Engineering Company, Jeddah, KSA. 2,3,5-Triphenyltetrazolium chloride was obtained from Gold Biotechnology, USA.

Peripheral blood samples are collected and dispatched to the clinical analysis laboratory for a comprehensive panel of tests. These tests include a complete blood count (flow cytometry and impedance – DXH 800), urea (urease with GLDH), creatinine (kinetic – IFCC), sodium, calcium, phosphate, and potassium (Selective Electrode), venous blood gases (Sensor-OMNI), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (kinetic – IFCC), and alkaline phosphatase (Abcam, Cambridge, UK).
To facilitate enzyme immunoassays, blood samples are collected and centrifuged at 3,500 rpm for 10 minutes. Plasma aliquots are cryopreserved in liquid nitrogen for subsequent measurements of N-terminal pro-B natriuretic peptide (NT-proBNP, Human pro-BNP Georgia, USA), fibroblast growth factor-23 (FGF-23; Abcam, Cambridge, UK), and parathyroid hormone (Sigma-Aldrich, San Luis, Missouri, EUA), α-Klotho (Quidel, San Diego, USA), and MILLIPLEX MAP Human Bone Magnetic Bead Panel - Bone Metabolism Multiplex Assay (Merck, Darmstadt, Germany). These quantitative measurements are performed in accordance with the manufacturer’s recommendations, and the absorbance is determined using a microplate reader (Biotek EPOCH 2).

LC3b was taken as a marker for autophagy. Resected thigh muscles were coated with paraffin and 4–6 μm sections were diced. Later samples were deparaffinised and rehydrated for coating with selected antibodies. BSA was used as a blocking agent and LC3 (Abcam) antibody was poured as primary antibody and incubated. After incubation with alkaline phosphatase (Abcam), the secondary antibody, substrate was provided and then counter staining with Mayer’s hematoxylin (Merck) was done and slides were observed under optical microscope at magnification of ×40.

The sections were deparaffinized and treated with Antigen Unmasking Solution (Vector, Burlingame, CA, USA) for 30 min at 100 °C. Subsequently, these sections were washed with phosphate buffered saline (PBS), and endogenous peroxidase activity was blocked by incubation in methanol containing 0.3% H₂O₂ for 30 min. After blocking with 2% horse serum (Vector) for half an hour, the sections were incubated overnight with primary antibodies against osteoprotegerin (Abcam, Cambridge, UK), alkaline phosphatase (GeneTex), angiogenin (Abcam), and osteopontin (Abcam). The sections were then incubated with biotinylated anti-rabbit IgG antibody (Vector) for 60 min. The ABC complex system (Vector) was used to subsequently develop peroxidase via incubation in a VIP solution (Vector) to visualize the peroxidase label. Images were obtained with a microscope equipped with a Charge-coupled Device (CCD) camera (AxioCam HRC, Zeiss, Jena, Germany) to detect the signal.

The committed MSCs were cultured with different concentrations of PBCA-Sr NPs for 7 days. The cells were lysed using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA) and centrifuged at 12,000 rpm at 4 °C for 15 min. Protein concentration was measured by Bio-Rad protein assay reagent. Protein separation was performed by using SDS polycarylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membrane. Blocking of non-specific binding was achieved by placing the PVDF membrane in a 1% BSA blocking solution and stained with antibodies specific for osteocalcin (OCN, 1:1000, Santa Cruz Biotechnology), alkaline phosphatase (1:1000, Abcam, Cambridge, UK) or β-actin (1:10000, Santa Cruz), gently shaken in room temperature for 1 h, then washed 3 times for 15 min with washing buffer (10 mM Tris-base, pH 7.5, 100 mM NaCl 0.1% Tween 20). Secondary antibody diluted in peroxidase-containing blocking buffer is added, incubated for 1 h, then, washed 3 times for 15 min with washing buffer. The PVDF membrane was analyzed by chemiluminescent-base detection system for detection of protein expression.