Inform automated image analysis software

Opal mIF staining was performed by the UCLA Translational Pathology Core Laboratory based on tyramide signal amplification (TSA). The Opal Polaris 7-Color Automation IHC Kit (Cat#NEL871001KT, Akoya Biosciences, Marlborough, MA, USA) was used with ER2 30 min retrieval and 1 h room temperature incubation. Staining was performed consecutively using each of the following antibodies: Paired Box 8 (PAX8, prediluted rabbit polyclonal antibody 363A-18, Cell Marque, Rocklin, CA, USA), calcyphosine (CAPS, Atlas Antibodies Cat#HPA043520, RRID:AB 10964138, 1:1000 dilution, Sigma-Aldrich, St. Louis, MO, USA), and tubulin beta 4 (TUBB4, Sigma-Aldrich, St. Louis, MO, USA), monoclonal anti-acetylated tubulin T7451, 1:500 dilution), and 4′,6-diamidino-2-phenylindole (DAPI) (Cat# SKU FP1490, Akoya Biosciences, Marlborough, MA, USA). The slides were scanned at 20X with the Vectra Polaris scanner (Akoya Biosciences, Marlborough, MA, USA) and data from the multispectral camera were analyzed using the imaging InForm automated image analysis software (Akoya Biosciences, Marlborough, MA, USA). Images were scored at 10X magnification using Phenochart whole slide viewer (Akoya Biosciences, Marlborough, MA, USA).

Multispectral fluorescence staining was performed using the Opal 6-Plex Detection kit (Akoya Biosciences) using FFPE tissue sections. Slides were baked at 60°C for 15 minutes and deparaffinized with the Leica Bond Dewax solution (Leica Biosystems), followed by heat-based antigen retrieval using Bond Epitope Retrieval Solution 1 (Leica Biosystems) for 30 minutes. Using the Leica Bond Rx Automated Stainer (Leica Biosystems), slides were incubated with primary antibodies followed by the appropriate secondary horseradish peroxidase–conjugated polymer. Incubation was next performed with a unique Opal dye permitting fluorophore covalent bonding to the horseradish polymer. Heat-based retrieval with Bond Epitope Retrieval 1 (Leica Biosystems) was finally performed for 20 minutes. Slides were subjected to sequential rounds of staining. Primary antibodies, concentrations, and associated fluorophores are detailed in Supplemental Table 9. Sections were counterstained with Spectral DAPI and mounted with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific). Images were acquired using the Vectra3 microscope (Akoya Biosciences) and Phenochart Whole Slide Viewer (Akoya Biosciences). Postacquisition image adjustments were performed using InForm Automated Image Analysis Software (Akoya Biosciences) and Fiji (74 (link)).

Multispectral fluorescence staining was performed using the Opal 6-Plex Detection kit (Akoya Biosciences, Marlborough, MA) using formalin-fixed, paraffin embedded tissue sections. Slides were baked at 60 °C for 15 min and deparaffinized with the Leica Bond Dewax solution (Leica Biosystems, Deer Park, IL), followed by heat-based antigen retrieval using Bond Epitope Retrieval Solution 1 (Leica Biosystems) for 30 min. Using the Leica Bond Rx^™ Automated Stainer (Leica Biosystems), slides were incubated with primary antibodies followed by the appropriate secondary horseradish peroxidase-conjugated polymer. Incubation was next performed with a unique Opal dye permitting fluorophore covalent bonding to the horseradish polymer. Heat-based retrieval with Bond Epitope Retrieval 1 (Leica Biosystems) was finally performed for 20 min. Slides were subjected to sequential rounds of staining. Primary antibodies, concentrations, and associated fluorophores are detailed in Supplemental Table 9. Sections were counterstained with Spectral DAPI and mounted with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific). Images were acquired using the Vectra3 microscope (Akoya Biosciences) and Phenochart Whole Slide Viewer (Akoya Biosciences). Post-acquisition image adjustments were performed using InForm Automated Image Analysis Software (Akoya Biosciences) and Fiji (76 (link)).