Bz x 810 digital microscope

The constructs were collected and fixed with buffered 4% paraformaldehyde (PFA) overnight. After equilibration in 15% and 30% sucrose solutions, the samples were embedded in OCT^® compound (Thermo Fisher Scientific, Waltham, MA, USA), and cryosectioned at 10 µm thickness. Before staining, the sections were washed in PBS. To check the distribution of collagen, the slides were then immersed in Weigert’s Hematoxylin, Herovici’s working solution, and 1% acetic acid, respectively. Finally, the slides were dehydrated through a series of graded ethanol baths and cleared in a xylene bath. To visualize the expression of α-smooth muscle actin, sections were first blocked in 8% FBS buffer for 1 h, then incubated with α-smooth muscle actin monoclonal antibody (Thermo Fisher Scientific, Waltham, MA, USA, 14-9760-82) overnight at 4 °C. After washing with PBS 3 times, the slides were incubated with goat anti-mouse secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA, A-11029). Sections were imaged using the BZ-X 810 digital microscope (Keyence, Osaka, Japan).