Goat serum

Cells seeded on the scaffolds were allowed to grow for 24 h followed by washing with PBS, and were subsequently fixed with 4% paraformaldehyde for 10 min. The fixed cells were then permeabilised for 20 min using 0.5% Triton X-100 (Scharlau, Barcelona, Spain) before blocking by immersion in 10% goat serum (Elabscience, Houston, TX, USA) for 1 h at 37 °C. HDF was incubated with the primary antibodies, rabbit anti-human collagen I antibody (1:300 dilution; Abcam, Cambridge, MA, USA) and mouse anti-alpha smooth muscle antibody (1:500 dilution; Abcam, Cambridge, MA, USA) overnight at 4 °C, followed by secondary antibody incubation with Alexa Fluor 647 goat anti-rabbit (Abcam) and Alexa Fluor 488 goat anti-mouse antibodies (Abcam), both diluted to 1:1000, for 2 h at 37 °C. The cells were counterstained with DAPI (Thermo Fisher, Waltham, MA, USA) for 15 min to visualise the nuclei. Between each post-fixing step, the samples were washed with PBS containing 0.1% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA). Images were captured using an A1R confocal laser scanning microscope (Nikon, Minato City, Japan).

After the cells were fixed with 4% paraformaldehyde for 15 minutes, they were washed with 0.01 mol/L PBS buffer solution for 3 times and then sealed with 10% goat serum + 0.03% Triton X-100 blocking solution (Elabscience, Wuhan, China) at room temperature for 2 h, and then, a primary antibody (SOD1, Abcam, Cambridge, MA, USA, Rabbit, 1 : 3000) was added, placed in a refrigerator at 4°C overnight, and then washed with 0.01 mol/L PBS buffer solution. The sheep anti-mouse secondary antibody (Jian Cheng, Nanjing, China) was added, incubated at room temperature for 2 h, and then washed with 0.01 mol/L PBS buffer solution. The cell nucleus was stained by 4′,6-diamidino-2-phenylindole (DAPI) (Jian Cheng, Nanjing, China) in the dark, and antifade solution (Jian Cheng, Nanjing, China) was added to the film and stored at 4°C for observation under fluorescence microscope (Keyence, Shanghai, China).