OMAP-16 Organ Mapping Antibody Panel (OMAP) for Multiplexed Antibody-Based Imaging of Human Lung with CODEX, v1.0

OMAP-16 was designed for Multiplexed Immunofluorescent (MxIF) imaging of human FFPE lung sections utilizing Akoya’s<br>Phenocycler-Fusion platform as described dx.doi.org/10.17504/protocols.io.6qpvr38dpvmk/v2. The panel contains<br>34-antibodies and the nuclear marker DAPI for image alignment and nuclear segmentation. OMAP-16 provides spatial<br>context for airway, vasculature, and alveolar features and identifies 35 of 72 key epithelial, endothelial, and immune<br>cell types in the ASCT+B lung table v1.2 (https://hubmapconsortium.github.io/ccf-releases/v1.2/docs/asct-b/lung.html).<br>OMAP-16 does not identify Cap1 and Cap2 endothelium, eosinophils/basophils, fibroblast/pericytes, neuroendocrine<br>cells, ciliated cells, chondrocytes, NK cells as Ab-Barcode conjugates targeting specific markers for these cell types<br>are in development. For select images cell segmentation based on DAPI stained nuclei was performed utilizing the<br>respective StarDist extension in QuPATH 0.3.2. Vascular smooth muscle and airway epithelial features are annotated<br>and classified by thresholding the ACTA2 and PanCK signals by utilizing the Pixel classifier in QuPath as<br>described. Object data is then exported as a GeoJSON file. Details regarding antigen retrieval in AR9 Buffer (Akoya<br>Biosciences) at high pressure (110C) 20’ and MxIF imaging lung section can be found in the protocol cited above. The<br>antibody validation protocol can be found here dx.doi.org/10.17504/protocols.io.36wgq3b9olk5/v1. Protein targets were<br>detected with antibody barcode conjugates obtained directly from Akoya Biosciences, or via custom conjugation of<br>commercially available antibodies utilizing antibody conjugation kit (Cat# 232195, Akoya). When imaging proximal lung<br>tissue containing large airway and vascular features, antibodies directed against extracellular matrix and vascular<br>smooth muscle should be imaged within the first five cycles to mitigate potential loss of feature integrity during<br>later cycles.