2.2. Immunofluorescence

OVCAR-3 cells were cultured at 50% confluence in Lab-Tek^® 3-well chamber slide for 24 h before fixation for 10 min with 4% paraformaldehyde. The cells were then washed with PBS. Non-specific binding sites were blocked using 1% BSA in PBS-tween (0.1%). Cells were then incubated with primary antibodies, anti-HLA-G (rabbit purchased from Epigentek, Farmingdale, NY, USA) and anti-PD-L1 (mouse purchased from Proteintech, Rosemont, IL, USA), respectively, for 3 h. Then, cells were washed with PBS-Tween (0.1%) and incubated with secondary antibodies (anti-rabbit AF488 and anti-mouse AF594; Invitrogen, Waltham, MA, USA) for 1 h. The incubations were performed at room temperature with mild agitation. The slides were then washed and mounted with DAPI containing aqueous mounting medium by Vectashield^® for images to be taken using ZEISS LSM 900 confocal microscope.