Quantifying Cell Proliferation via Ki-67

Cell proliferation was assessed using Ki-67 immunostaining as described previously (10 (link), 32 (link), 34 (link)). Briefly, cells were washed with PBS, fixed in 4% paraformaldehyde/PBS, and permeabilized using Triton X-100/PBS solution. Then cells were incubated in blocking solution (2% BSA/PBS), followed by overnight incubation with primary anti-Ki-67 antibody (#9129, Cell Signaling, Danvers, MA, United States) in blocking solution. Next day, the slides were washed with PBS, followed by incubation with secondary chicken anti-rabbit IgG (H + L) Alexa Fluor 594 antibody (#A-21442, Invitrogen, Waltham, MA, United States) and 4′,6-diamidino-2-phenylindole (DAPI) to detect nuclei. Images were taken using a Keyence BZ-X800 microscope.