Prolong gold antifade mountant with dapi

After separation, the cells collected from all outlets were labeled using immunofluorescence staining to identify tumor cells and WBCs. Specifically, cells were first obtained by the centrifugation of collected samples and coated onto Polysine adhesion slides (P4981, Thermo Fisher Scientific) at room temperature. The adherent cells were then fixed with a −20 °C methanol solution for 5 min. After incubation in PBS buffer with 10% normal goat serum (Abcam), the tumor cells were stained with fluorescein isothiocyanate (FITC)-conjugated Pan-CK monoclonal antibody (Thermo Fisher Scientific), while the WBCs were stained with allophycocyanin (APC)-conjugated CD45 antibody (BioLegend) at 4 °C overnight. The fluorescently labeled cells were mounted using Prolong Gold Antifade Mountant with DAPI (Sigma‒Aldrich). Cells that were positive for Pan-CK and DAPI but negative for CD45 were identified as tumor cells, while cells that were positive for CD45 and DAPI but negative for Pan-CK were identified as WBCs.

HuR-overexpressing and control 786–0 cells were plated on cover slips and cultured for 24 h. Cells were fixed by 4% PFA for 15 min, permeabilized by 0.25% Triton X-100 for 15 min and blocked with 5% BSA/PBS at room temperature for 1 h. Primary antibody (goat anti-PD-L1, PA5–18337, Thermo Fisher) was then incubated in humidified chamber at room temperature for 1 h, followed by rigorous wash with PBST. Alexa Fluor 488-labelled secondary antibody (donkey anti-goat, A32814, Thermo Fisher) was incubated for another hour in the dark. Immediately after PBST wash, the cover slips were mounted with ProLong^™ Gold Antifade Mountant with DAPI (Sigma-Aldrich). The images were captured under confocal microscope (LSM800, Carl Zeiss, Oberkochen, Germany).