Mouse monoclonal anti osteocalcin

For immunocytochemical analysis of the osteoblast cells cultured on different pore size PCL scaffolds (tissue culture plate acted as the control group) for 14 and 30 days, cells were fixed with 4% paraformaldehyde in PBS (Polysciences, Warrington, PA, USA) for 30 min at room temperature then gently rinsed with PBS. The cell membranes were then permeabilized and blocked with a protein blocker solution (1% BSA, 22.52 mg Glycine in 0.1% Tween 20 in PBS), Sigma Aldrich) for 30 min. After washing, the cells were incubated in the following diluted primary antibodies at 4 °C overnight: mouse monoclonal anti-Collagen IA (1:250, SantaCruz Biotechnology, USA), rabbit polyclonal anti-Collagen III (1:100, abcam, Australia), mouse monoclonal anti-Osteocalcin (1:200, abcam, Austrailia).
The cells were rinsed in PBS (three times, 5 min per wash) and incubated in the appropriate secondary antibody i.e. Alexa Fluor 488-conjugated goat anti-rabbit (1:200, abcam, Australia) or F (ab^`)2-Goat anti-Mouse IgG FITC (1:200, ThermoFisher Scientific, USA) at room temperature in the dark for 1 h. Cell nuclei were stained using 40, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA) in PBS (1:1000) for 30 min. The samples were mounted onto glass slides for visualisation using a fluorescence microscope (Nikon, Eclipse- Ti, U.S.A).

To further evaluate the late osteogenic differentiation as well as the maturation of extracellular matrix (ECM), collagen-I and osteocalcin immunocytochemical stainings were conducted, as described in detail previously[22 (link)]. Primary antibodies mouse monoclonal anti-collagen-I (dilution 1:2000) and mouse monoclonal anti-osteocalcin (dilution 1:100) (Abcam, Cambridge, UK) and secondary antibody donkey anti-mouse Alexa fluor 488 IgG (Invitrogen, Thermo Fisher Scientific; dilution 1:800) were used. Secondary antibody was applied together with actin-staining phalloidin-TRITC (Sigma-Aldrich, dilution 1:500). The nuclei were stained with DAPI (Molecular Probes, Thermo Fisher Scientific; dilution 1:2000) during the wash steps after the secondary antibody treatment.
In addition to the collagen-I and osteocalcin stainings, the structure of the actin cytoskeleton was visualized with phalloidin staining after 7 days of culture. For this, the samples were fixed, permeabilized and blocked, and the phalloidin and DAPI stainings were conducted as described in ref.[22 (link)], but with the omission of the antibody treatments. All the samples were imaged with the aforementioned microscope.