Rabbit anti factor 8

To validate the EC-like cells, Factor VIII expression was assessed using an immunohistochemistry assay as previously described (31 (link)). Cells were fixed using 4% paraformaldehyde for 20 min at room temperature, treated with 0.3% H₂O₂ for 5 min at room temperature, permeabilized using 0.5% Triton X-100 and blocked using goat serum (Beyotime Institute of Biotechnology, Haimen, China) for 10 min at room temperature. The cells were incubated with rabbit anti-Factor VIII (1:200; BIOSS, Beijing, China; no. bs-0434R) for 4 h at 37°C. Following incubation with biotinylated goat anti rabbit IgG (1:200; Wuhan Boster Biological Technology, Ltd., Wuhan, China; BA1006) for 30 min at room temperature, the cells were treated with a streptavidin-biotin complex for 20 min at 37°C. The final immunoreactions were conducted using 3,3′-Diaminobenzidine for 5 min at room temperature according to the manufacturer's protocol (Wuhan Boster Biological Technology, Ltd.). Finally, the slides were mounted with neutral balsam (Origene Technologies, Inc., Beijing, China) and observed under an inverted microscope at ×200 magnification (Olympus Corp., Tokyo, Japan).