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).
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).