Cy3 conjugated goat anti mouse secondary antibody

After 7 days in culture, immunofluorescence staining of collagen-I was performed to observe the distribution of this protein in the cells on aligned and random scaffolds. The cell-seeded scaffolds were rinsed with PBS twice, fixed with 4% paraformaldehyde for 15 min and perforated by methanol at −20 °C for 5 min. The non-specific binding was blocked by 4% bovine serum albumin (BSA) for 30 min at room temperature. Subsequently, the samples were incubated with a monoclonal anti-collagen-I antibody (Abcam, 90395) at a dilution of 1:200 at 4 °C overnight. After washing in PBS three times, the samples were further incubated with Cy3-conjugated goat-anti-mouse secondary antibody (1:300, Invitrogen, A10521) for 1 h. In addition, nuclei were stained with DAPI for 5 min. After thoroughly washing in PBS, the collagen-I distribution was visualised under a fluorescence microscope (Zeiss Axiovert 200, Carl Zeiss Inc., Thornwood, NY).