Primary antibodies included those to CD8, CD68, FoxP3, pan-CK (clone AE1/3), PD-1, and PD-L1 (table 1). All sites used primary antibodies from the same lot. For CD8 and CK: Akoya’s Opal Polymer anti-mouse and -rabbit HRP (1:5, ARH1001EA) was used for secondary detection. Leica Biosystems PowerVision Poly-HRP antimouse was used for FoxP3 and CD68 (50%, PV6114, Leica Biosystems) and Poly-HRP anti-rabbit was used for PD-1 and PD-L1 (50%, PV6119, Leica). Each site received an Opal 7-color Automated IHC Detection Kit (NEL821001KT, Akoya Biosciences, Marlborough, Massachusetts, USA) containing the following TSA fluorophores: Opal 520, Opal 540, Opal 570, Opal 620, Opal 650, Opal 690, and spectral DAPI. All fluorophores and DAPI were prepared according to manufacturer guidelines.

Final, optimized six-plex mIF assay conditions for characterizing the PD-1/PD-L1 axis

*All antibodies were diluted using Akoya’s antibody diluent/blocking buffer.

†This antibody is not purified and is supplied as crude tissue culture supernatant. As such, the antibody concentration is not available.

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