Multiplex Immunohistochemistry for Immune Profiling

Details on mutational analysis and immunohistochemical (IHC) staining for PD-L1 expression and CD8 infiltration was previously reported [9 (link)]. Double staining CD3 (yellow) followed by CD56 (purple) of whole slide sections prepared from FFPE resection specimens was performed on a Discovery Ultra autostainer. Slides were deparaffinised in the instrument and heat-induced antigen retrieval was carried out using Cell Conditioning 1 (CC1, Ventana Medical Systems) for 32 min at 95 °C. The CD3 was detected in the first sequence using clone SP7 (1/100 dilution, 32 min at 37 °C, ThermoScientific). CD3 bound antibody was visualized using Anti-Rabbit NP (Ventana Medical systems) for 12 min at 37 °C followed by Anti-NP AP (Ventana Medical systems) for 12 min at 37 °C, followed by the Discovery Yellow detection kit (Ventana Medical Systems). In the second sequence of the double staining procedure CD56 was detected using clone MRQ-42 (1:2000 dilution, 32 min at 37 °C, Cell Marque). CD56 was visualized using Anti-Rabbit HQ (Ventana Medical systems) for 12 min at 37 °C followed by Anti-HQ HRP (Ventana Medical systems) for 12 min at 37 °C, followed by the Discovery Purple Detection Kit (Ventana Medical Systems). Slides were counterstained with Hematoxylin and Bluing Reagent (Ventana Medical Systems).