Opal multiplex fihc kit

mIHC/IF was performed (n = 34) using an Opal Multiplex fIHC kit (Akoya Bioscience, Menlo Park, CA, USA), as previously described^{5 (link)}. All cases profiled were also correspondingly profiled on the NanoString PanCancer IO360 panel and included in a combined analysis. Briefly, slides were labeled with PD-L1, CD68, CD8, Foxp3, CD15, and ERG, followed by appropriate secondary antibodies (detailed protocol previously reported^{46 (link)}). Ten images of viable tumor regions that were selected randomly by pathologists were acquired for each case using a Vectra 3 pathology imaging system microscope (Akoya Bioscience, Menlo Park, CA, USA) then analyzed and scored by a pathologist. The number of immune cells scored were normalized against ERG+ (tumor) cells and log2 transformed prior to correlating with NanoString data.

mIHC/IF was performed on FFPE biopsy tissue using Opal Multiplex fIHC kit (Akoya Biosciences, Menlo Park, CA, USA)^{51 (link)}. In brief, 4 um FFPE tissue sections were semi-automatically processed using Leica Bond Max autostainer (Leica Biosystems, Wetzlar, Germany). The slides were stained using 6 primary antibodies (CD8, FOXP3, PD-1, CTLA-4, TCF1 and CD39; summarized in Supplementary Table 5), polymeric HRP-conjugated secondary antibodies from BOND Polymer Refine Detection kit (Cat DS9800; Leica Biosystems, Newcastle, UK), and Opal fluorophore-conjugated tyramide signal amplification (1:100 dilution; Cat #NEL797001KT; Akoya Biosciences, Marlborough, MA, USA). Mounted slides were scanned using Vectra 3 pathology imaging system microscope (Akoya Biosciences). Images were analyzed using inform software (v2.4.6; Akoya Biosciences). Statistical analysis and visualization were performed using GraphPad Prism (v8.0.0; GraphPad Software, Inc., San Diego, CA, USA).

mIHC was performed using an Opal Multiplex fIHC kit (Akoya Biosciences, California). FFPE tissue sections were cut onto Bond Plus slides (Leica Biosystems, Richmond) and heated at 60 °C for 20 min. The tissue slides were then subjected to deparaffinization, rehydration and heat-induced epitope retrieval using a Leica Bond Max autostainer (Leica Biosystems, Melbourne) before endogenous peroxidase blocking (Leica Biosystems, Newcastle). Next, the slides were incubated with primary antibodies followed by incubation with polymeric HRP-conjugated secondary antibodies (Leica Biosystems, Newcastle) (Supplementary Table 3). Then, the samples were incubated with Opal fluorophore-conjugated tyramide signal amplification (TSA) (Akoya Biosciences, California) at a 1:100 dilution. The slides were rinsed with wash buffer (BOND Wash Solution 10X Concentrate) after each step. Following TSA deposition, the slides were again subjected to heat-induced epitope retrieval to strip the tissue-bound primary/secondary antibody complexes prior to further labeling. These steps were repeated until the samples were labeled with all six markers and spectral DAPI (Akoya Biosciences, California) at a 1:10 dilution. Finally, the slides were mounted in ProLong Diamond Anti-fade Mountant (Molecular Probes, Life Technologies, USA) and developed in the dark at room temperature for 24 h.