Immunofluorescence Staining of Cell Markers

The cells were fixed in 4% paraformaldehyde at 25 °C for 15 min. After washing with phosphate-buffered saline (PBS), the cells were blocked with blocking buffer (5% goat serum and 0.3% Triton X-100 in PBS) at room temperature for 1 h. After removing the buffer, the cells were incubated with primary antibodies (anti-Ki67, #12202; anti-active-β-catenin, #05-665, Merck Millipore, Billerica, MA, USA; and anti-TOMM20, ab186735, Abcam, Cambridge, UK) at 4 °C overnight. After washing with PBS, the cells were incubated with secondary antibodies (Alexa Fluor 555 anti-rabbit IgG, Alexa Fluor 488 anti-rabbit IgG, or Alexa Fluor 555 anti-mouse IgG) at 25 °C for 2 h. Then, the cells were washed and stained with Hoechst 33342 at room temperature for 20 min and observed under a fluorescence microscope (BZ-X800, Keyence, Osaka, Japan) [3 (link)].