Differentiation of C2C12 cells and immunofluorescence staining

C2C12 cells were cultured in the hydrogels at a cell density of 3000 cells per well in the above-mentioned µ-slide. When cells achieved 50% confluence, the 10% FBS DMEM medium was replaced with low-glucose DMEM supplemented with 2.5% horse serum and 1% penicillin/streptomycin in order to induce cell differentiation. Then, 72 and 168 h after the initial setup of culture in differentiation medium, the cells were fixed with 4% PFA for 1 h. Cells were then permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, T9284) for 20 min at 25 °C, followed by blocking with 3% BSA for 12 h at 4 °C. Subsequently, cells were incubated with phalloidin for 30 min or exposed to immunofluorescent staining. Therefore, samples were incubated with primary mouse anti-Cardiac Troponin T antibodies (Abcam, ab8295) diluted at a ratio of 1:400 for 2 h. Secondary antibodies, anti-mouse Alexa Fluor 647 (Invitrogen, A-21235) were applied at a ratio of 1:500 for 1 h and nuclei were counterstained with Hoechst 33342 (1:4000). Then, samples were imaged with the confocal laser scanning microscope mentioned above.