Immunohistochemical Staining Protocol for PD-L1

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.