Haematoxylin 2 and bluing reagent

Specimens were sectioned at a thickness of 3 μm and stained on positively charged glass slides stored at 4°C within 3 days after sectioning. Deparaffinization, rehydration, and antigen retrieval were performed by CC1 (prediluted; pH 8.0) antigen retrieval solution (ref. 950–124, Ventana Medical Systems, Inc.), performed on the VENTANA BenchMark ULTRA automated slide stainer for 64 minutes at 100°C. Specimens were incubated with primary mouse anti–PD-L1 monoclonal antibody (ref. M365329; Dako) using a concentration of 1:50 for 32 minutes at 37°C, followed by visualization with the OptiView DAB IHC Detection Kit (Ventana) and OptiView Amplification Kit (Ventana) for 12 minutes. The specimens were then counterstained with haematoxylin II and bluing reagent (Ventana) and coverslipped. Each IHC run contained a positive control (on-slide tonsil tissue) and a negative antibody control (buffer, no primary antibody) (http://dx.doi.org/10.17504/protocols.io.ixacfie).

Immunohistochemical protocol was adapted from Ilie et al.^{58 (link)}. Specimens were sectioned at a thickness of 3 μm and stained on positively charged glass slides. Deparaffinization, rehydration, and antigen retrieval were performed by CC1 (prediluted; pH 8.0) antigen retrieval solution (Ventana Medical Systems, Inc.), performed on the VENTANA BenchMark ULTRA automated slide stainer for 32 min at 100 °C. Specimens were incubated with primary antibodies as noted in Supplementary Table 1 followed by visualization with the OptiView DAB IHC Detection Kit (Ventana) and OptiView Amplification Kit (Ventana) for 12 min for PD-L1 detection. The specimens were then counterstained with haematoxylin II and bluing reagent (Ventana) and coverslipped. Each IHC run contained a positive control (on-slide placenta tissue for PD-L1). Morphological characteristics and size of the nucleus were taken into account to estimate the labeling of fibroblasts by vimentin antibodies.