Biotinylated goat anti rabbit igg

The cells on coverslips were incubated with rabbit anti-microtubule-associated protein 2 antibody (1:100; Abcam, Cambridge, UK) at 4°C overnight. Cells were then incubated with biotinylated goat anti-rabbit IgG (1:100; Wuhan Boster Biological Technology, Wuhan, China) secondary antibody at 37°C for 1 hour, and the cells were then incubated with streptavidin biotin complex-Cy3 (1:100; Wuhan Boster Biological Technology) at 37°C for 30 minutes. A negative control was carried out using the same procedures without primary antibody. The cells on coverslips were visualized with green fluorescence at 488 nm, and microtubule-associated protein 2-positive cells were visualized with red fluorescence at 568 nm under a fluorescence microscope (Olympus, Sunnyvale, CA, USA). Confocal images (double-fluorescent cells) were yellow fluorescent and were obtained with a laser scanning confocal microscope imaging system (Olympus). Values of microtubule-associated protein 2-positive cell ratio were calculated by the number of cells with yellow fluorescence/the number of cells with green fluorescence × 100%.

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).

Goat anti-pig IgA (1:100; cat. no. ab112639) and rabbit anti-pig CD3 (SP7) (1:100; cat. no. ab16669) monoclonal antibodies were purchased from Abcam. The streptavidin-biotin complex (SABC) kit (Wuhan Boster Biological Technology, Ltd), including biotinylated goat anti-rabbit IgG (cat. no. sa1022), biotinylated rabbit anti-goat IgG antibodies (cat. no. sa1023) and horseradish peroxidase (HRP)-labeled SABC, was purchased from Boster Biological Technology. The 3,3-diaminobenzidine (DAB) HRP Color Development kit (cat. no. ar1022) was also purchased from Boster Biological Technology.