2.1. Immunostaining of Human Colorectal Cancer Cell Lines

Human colorectal cancer cell lines (HCT116, HT29, and DLD-1) (obtained from European collection of cell cultures, Culture Collections of Public Health England, UK; Depositor: All cell lines were obtained from the American Type Culture Collection) were cultured at 37 °C under 5% CO₂ for 24 h in an RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) with 10% fetal bovine serum. Then, the slide was fixed in 4% paraformaldehyde (4%PFA) diluted by phosphate-buffered solution for 1 h at room temperature (RT). The slide was rinsed 3 times for 5 min in phosphate-buffered containing 0.3% Triton-X (PBT), then incubated in 3% FBS for 30 min at RT to block binding nonspecific antibodies, and stained overnight at 4 °C using an anti-PROK1 mAb prepared by our department [17].