Immunodetection with confocal microscopy

Colon cancer cell lines HCT116, DLD1, SW480, HT29, and RKO were cultured on a chamber slide (NUNC) and the cells were grown until they reached 70–80% confluence. Then, a 4% paraformaldehyde fixation solution was poured onto the cells and incubated for 30 min at room temperature. After being rinsed and washed with PBS, the cells were permeabilized with PBS containing 0.01% Triton X-100 for 10 min. After being blocked with PBS containing 0.01% Triton X-100 and 5% BSA for 45 min at room temperature, the cells were incubated with a rabbit anti-human 4-1BBL primary antibody for 30 min. Then, after being twice washed with PBS containing 0.01% Triton X-100, the cells were incubated with DyLight 633 conjugated goat anti-rabbit secondary antibody (ImmunoReagents, Inc., Raleigh, NC, USA) for 30 min at room temperature. After they were twice washed with PBS containing 0.01% Triton X-100, the cells were incubated with mouse anti-human E-Cadherin (Abcam plc, Cambridge, UK) or mouse anti-human β-catenin (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) primary antibody for 30 min. Then, after being washed twice, the cells were stained with DyLight 488-conjugated goat anti-mouse secondary antibody (Thermo Fisher Scientific). The cells were then washed twice and the nuclei were stained with DAPI for 10 s. After being washed twice, the cells were covered with a coverslip by adding 50 μL of anti-fade medium and subsequently sealed. Normal rabbit serum (Novus Biologicals) was used as the control for rabbit anti-human 4-1BBL staining. All images were observed using confocal laser scanning microscopy (Leica Microsystems).