Automated mIF was performed as previously described (Giraldo et al. 2018 (link); Davis et al. 2020 (link)). Briefly, slides were heated and dewaxed to remove any paraffin. Antigen retrieval was performed using ER2 followed by washing steps. Nonspecific staining was blocked using Blocking/Ab Diluent (Akoya Biosciences) followed by the first primary antibody (see position 1 in Supplemental Table S3 ). The corresponding polymer was applied followed by the tyramide signal amplification dye (Opal Automation Multiplex IHC Kit; Akoya Biosciences). Slides were heated to strip the primary antibody and polymer, washed, and blocked again. The process was repeated for positions 2–6. After the last step of antibody striping, the slides were stained for DAPI and coverslipped using ProLong Diamond Antifade Mountant (Life Technologies).
Slides were scanned using the Vectra Polaris Quantitative Pathology Imaging System (Akoya Biosciences). A 10× (1 µm/px) whole-slide scan was acquired and used as a guide to select 20 high power field (HPF) for 20× image acquisition. These 20× HPF images were processed in Inform software (Akoya Biosciences) and exported to images with QPTIFF format.
Slides were scanned using the Vectra Polaris Quantitative Pathology Imaging System (Akoya Biosciences). A 10× (1 µm/px) whole-slide scan was acquired and used as a guide to select 20 high power field (HPF) for 20× image acquisition. These 20× HPF images were processed in Inform software (Akoya Biosciences) and exported to images with QPTIFF format.
