Immunohistochemical Analysis of Immune Checkpoint Markers

For immunohistochemical staining, formalin-fixed and paraffin-embedded tumour sections were pretreated with pH 9 Tris/EDTA buffer for 40 min at 95°C, quenched with 0.05% H₂O₂, and incubated overnight at 4°C with the following primary antibodies in Can Get Signal Immunostain Solution A (Toyobo, Osaka, Japan): PD-L1 polyclonal antibody (Invitrogen), PD-L2 polyclonal antibody (Invitrogen), and galectin-9 polyclonal antibody (Invitrogen). Following this, sections were incubated with horseradish peroxidase- (HRP-) conjugated goat anti-rabbit IgG polyclonal antibody (Nichirei Bioscience, Tokyo, Japan) for 30 min at room temperature. Signals were developed as a brown reaction product using peroxidase substrate 3,3′-diaminobenzidine (Nichirei Bioscience). The sections were counterstained with haematoxylin and examined under a BZ-8000 confocal microscope (Keyence, Osaka, Japan). Immunohistochemical staining was quantified using Hybrid cell count BZ-H3C software (Keyence) [16 (link)].