Af1019

Immunofluorescent staining was performed on 4 µm formalin-fixed, paraffin-embedded ovarian tumour sections. After deparaffinization, antigen retrieval was performed by boiling slides in sodium citrate buffer, and permeabilization was performed for nuclear antigens only using 0.25% v/v Triton X-100. Non-specific antibody binding was blocked with 5% BSA in PBS, and primary antibodies were applied and incubated overnight at 4 °C. Primary antibodies used were rabbit anti-wide-spectrum cytokeratin (#ab9377, 1:100, Abcam, Cambridge, UK), rabbit anti-Wilms tumor protein-1 (#ab89901, 1:100, Abcam), goat anti-PD-L1 (#AF1019, 1:125, R&D Systems, Minneapolis, MN, USA) and rabbit anti-p53 (#ab31333, 1:100, Abcam). Alexa Fluor® 594 or 647-conjugated goat anti-rabbit or donkey anti-goat secondary antibodies (1:1000, Abcam) were applied at 1:1000 for 1 h, and slides were mounted using ProLong™ Gold Antifade Mountant with DAPI (ThermoFisher Scientific). Fluorescence images were captured using the Cytation 3 imaging Multi-Mode Reader (BioTek) and processed using the Gen5™ software v 2.05 (BioTek).

Mouse tumors were excised either when they reached 10 mm or when they started to shrink following vaccine therapy. Specimens were fixed in 10% neutral buffered formalin (pH 6.8–7.2; Richard-Allan Scientific, Kalamazoo, Michigan, US) for paraffin embedding and sectioning. Five μm tissue sections were cut with a microtome, and sample processing and IF staining were performed as previously described using the following primary antibodies: CD3 rabbit anti-mouse mAb (1:100, ab16669, Abcam, Cambridge, Massachusetts, US) and PD-L1 goat anti-mouse polyclonal Ab (1:20, AF1019, R&D Systems, Minneapolis, Minnesota, US). Isotype-matched antibodies were used for negative controls. Sections were mounted with ProLong Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific, Halethorpe, Maryland, US).