The VENTANA PD-L1 (SP142) rabbit monoclonal primary antibody (Ventana Medical Systems Inc.) was optimized for use as a fully automated IHC assay on the BenchMark ULTRA (Ventana Medical Systems Inc., Tucson, AZ) staining platform using the OptiView DAB IHC Detection Kit and OptiView Amplification Kit (Ventana Medical Systems Inc., Tucson, AZ). The assay was optimized for detection of PD-L1 expression in NSCLC, where TC and IC are predictive8 (link) and in UC, where IC is predictive.11 (link) Parameters evaluated during optimization included antibody concentration, antibody diluent, multiple antigen retrieval methods, antibody incubation conditions, and counterstain conditions. In addition, signal amplification using the OptiView Amplification Kit (Ventana Medical Systems Inc.) was tested to evaluate its efficacy in visualizing IC staining. The optimal conditions for staining both TC and IC in NSCLC and UC tissues on the BenchMark ULTRA instrument are outlined in Table 1. Briefly, antigen retrieval was undertaken for 48 minutes, the primary antibody was applied for 16 minutes at 36°C, amplification was done for 8 minutes amplifier/8 minutes multimer, and samples were counterstained for 4 minutes with hematoxylin II and postcounterstained for 4 minutes.