Proteins of the differentiated SaOS-2 cells were extracted with a RIPA buffer (Tech & Innovation, Gangwon, Republic of Korea) containing protease inhibitors (Roche, Basel, Swiss) and separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated proteins were transferred onto a membrane (Bio-Rad, Hercules, CA, USA) and incubated with mouse and rabbit monoclonal anti-OCN, -RUNX family transcription factor 2 (Runx2), -Osterix and -ALP (Cell Signaling Technology; Danvers, MA, USA). After incubation with anti-mouse and rabbit IgG secondary antibody conjugated with horseradish peroxidase (Cell Signaling Technology), the immunoreactive bands were visualized by a chemiluminescence imaging system (GE Healthcare, Little Chalfont, UK) using enhanced chemiluminescence reagents. The band intensity was quantified using the Image J software (v1.4.3.x., U.S. National Institutes of Health, Bethesda, MD, USA). All experiments were performed in triplicate.
Anti ocn
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-OCN is a primary antibody product from Cell Signaling Technology. It is designed to detect Osteocalcin (OCN), a protein involved in bone mineralization and calcium ion homeostasis.
Automatically generated - may contain errors
Lab products found in correlation
Chemiluminescence imaging system, by GE Healthcare (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Alexa fluor 488, by Abcam (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Tcs sp8, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Blocking buffer, by Beyotime (1 mentions)
3 protocols using anti ocn
1
Western Blot Analysis of Osteogenic Markers
2
Osteogenic Differentiation of BM-MSCs
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After 7 days of osteogenic culture, the BM-MSCs cultured on different groups were fixed with paraformaldehyde (4 wt %) at 4 °C for 30 min. Subsequently, 0.2% Triton X-100 (Beyotime, China) was added to permeabilize cells and all the samples were sealed with Blocking Buffer (Beyotime, China). Afterwards, primary antibodies were added into each well (anti-OCN, 1:200, Cell Signaling Technology, USA) for the incubation at 4 °C overnight. The cells were then rinsed with PBS for 3 times, and the corresponding fluorescent secondary antibody (Alexa Fluor 488, ab150077, Abcam, UK) was added for cultivation in the dark for 1 h. All the samples were counterstained with DAPI (Beyotime, China), placed on a microscope slide with fluorescence anti-fade medium (Beyotime, China), and observed under a laser confocal microscope (TCS SP8, Leica, Germany).
3
In Vivo Bone Formation Capacity of Engineered hBMSCs
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The hBMSCs infected with lentivirus were induced in OM for 7 days before the in vivo experiments. Next, 5 × 106 cells were mixed with 7 mm × 5 mm × 2 mm Bio-Oss Collagen (Geistlich, GEWO GmbH, Baden-Baden, Germany), incubated for 1 h at 37 °C, and then implanted subcutaneously into the dorsal surface of 8-week-old BALB/c homozygous nude (nu/nu) female mice (n = 6 per group). The mice were randomized into 5 groups: shNC-hBMSCs/Collagen group, shMIR22HG-1-hBMSCs/Collagen group, shMIR22HG-2-hBMSCs/Collagen group, NC-hBMSCs/Collagen group and MIR22HG-hBMSCs/Collagen group. The implants were collected after 8 weeks and fixed with 4% formalin, decalcified, and embedded in paraffin wax. Sections (5 μm) were cut and stained with HE, Masson’s trichrome and immunohistochemical analysis. Immunohistochemical analysis was performed to detect the expression level of osteocalcin (OCN) (anti-OCN, Cell Signaling Technology).
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