Aperio digital at2 pathology system

Formalin-fixed paraffin-embedded tumour tissue was sliced into 4 μm full face sections and processed for 3′,3′-diaminobenzidine (DAB) immunohistochemistry using a Ventana Discovery Ultra (Roche, USA) by the HCB. Sections were labelled for T cell markers CD3 (2GV6; Roche), CD4 (SP35; Roche), CD8 (SP57; Roche), and PD-1 (NAT105; Sigma-Aldrich, USA). All steps from baking to chromogen addition were performed automatically by the instrument. Tissue sections were baked to slides and deparaffinized, and antigen retrieval then occurred at 95°C/pH 9 with a total incubation time of 24 minutes prior to the addition of the primary antibody. Addition of the primary antibody was followed by a 32-minute incubation at 36°C. For CD4 labelling only, the primary antibody was dispensed before the blocking step, resulting in a reduction of non-specific labelling in the tumour tissue. Slides were then incubated with secondary antibody – Anti-Rabbit HQ for CD3, CD4 and CD8, and Anti-Mouse HQ (Roche) for PD-1 – for 20 minutes at 36°C. Positive control (normal tonsil) and negative control (normal brain) tissues were included in each batch of slides to confirm the specificity of antibody labelling (Supplementary Figure 2). Slides were digitally scanned using the Aperio^™ Digital AT2 Pathology System (Leica Biosystems, Australia) for analysis.