To prepare specimens for multiplex IF staining, tissue sections were cut at 4 μm from formalin-fixed paraffin-embedded blocks. All sections on slides were deparaffinized using the Bond ER2 Leica Biosystems AR9640, followed by staining with the Leica Bond RX autostainer. The slides were stained with - CD3 (1:50 SP7 Abcam ab16669), CD16 (1:50 2H7 CellSignaling Technologies 88251S), CD32b (1:2000 NovusBiologicals NBP2–89364), and CD68 (1:400 PGM-1 DAKO). Antibody binding was visualized using secondary anti-mouse/anti-rabbit polymer HRP followed with TSA-Opal reagents (Akoya NEL811001KT). Tissue slides were incubated with DAPI as a counterstain and coverslipped with VectaShield mounting media (Vector Labs). Control tissue samples were stained for each marker as positive controls.
Whole slide digital images were captured with the PhenoImager HT platform and analyzed with QuPath software24 (link). Tissue samples from consecutive slides were stained by conventional H&E and scanned with the Leica SCN400F platform. H&E slides in conjunction with Cytokeratin staining were used to annotate tumor regions within the whole slide image. Cells were segmented, phenotyped, and enumerated using the QuPath object classification algorithm.
Whole slide digital images were captured with the PhenoImager HT platform and analyzed with QuPath software24 (link). Tissue samples from consecutive slides were stained by conventional H&E and scanned with the Leica SCN400F platform. H&E slides in conjunction with Cytokeratin staining were used to annotate tumor regions within the whole slide image. Cells were segmented, phenotyped, and enumerated using the QuPath object classification algorithm.