Vectra multispectral imaging platform

Multiplex immunofluorescence staining was performed on 5-micron thick formalin fixed paraffin embedded (FFPE) tissue sections as previously described using a Bond RX autostainer.11 12 (link) The target antigens, antibody clones, and dilutions for markers included in panels 1, 2, and 3 are listed in online supplemental table 3. Following staining, slides were manually counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (NucBlue Fixed Cell ReadyProbes Reagent, Invitrogen, Carlsbad, California), washed with deionized water, air-dried, and mounted with ProLong Diamond Antifade Mountant (Invitrogen). Image acquisition was performed using the Vectra multispectral imaging platform (PerkinElmer, Hopkinton, Massachusetts). Representative regions of interest were selected, and two to six fields of view (FOV) in these regions were acquired at 20× resolution as multispectral images. Once the FOV were spectrally unmixed, cell identification was performed using supervised machine learning algorithms within Inform V.2.3 (PerkinElmer). Thresholds for ‘positive’ staining were set under pathologist supervision for each case, then used to calculate phenotyped cell densities.