Immunocytochemistry Protocol for AGS Cells

AMOTL1 (HPA001196, Sigma), YAP1 (ab52771, Abcam), CTGF antibody (sc-14939, Santa Cruz), Ki67 (550609, BD Pharmingen), and cleaved-Caspase 3 (#9664, CST) were commercially available. The immunohistochemistry and the scoring of the results were performed as previously described [13 (link)]. For immunocytochemistry studies, AGS was cultured on coverslips in a six-well plate. After removal of the medium and three times of PBS washing, the cells were fixed with 4% paraformaldehyde at room temperature for 15 min. Followed by three times of PBS washing, the cells were then permeated with 0.1% Triton X-100 at room temperature for 15 min, and another PBS washing three times. Room-temperature blocking was done with 2% BSA for 45 min; then cells were incubated with the primary antibody (1:200) at 4 °C overnight. Again, after one time of PBS washing, the cells were incubated with goat anti-mouse IgG secondary antibody (Alexa Fluor 594, 1:400, Thermo Fisher Scientific) in the dark at room temperature for 1 h. After washing, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific). Images were captured with a microscope (Carl Zeiss Axio Imager 2, Oberkochen, Germany).