OMAP-25: Expanded Organ Mapping Antibody Panel (OMAP) for Multiplexed Antibody-Based Imaging of Human Lung with CODEX, v1.1

OMAP-25 was developed for Multiplexed Immunofluorescence (MxIF) imaging of human formalin-fixed paraffin-embedded<br>(FFPE) lung sections using the Akoya Biosciences Phenocycler-Fusion platform (Purkerson, Pryhuber, Colon, et al.<br>2025a). This panel includes 45 antibodies and the nuclear marker DAPI for image alignment and nuclear segmentation.<br>OMAP-25 provides spatial context for airway, vasculature, and alveolar structures, identifying approximately 40 out of<br>72 key epithelial, endothelial, and immune cell types defined in the ASCT+B lung table v1.2 (Pryhuber 2021). While the<br>panel offers broad coverage of lung cell types, it does not yet identify capillary type 1 and capillary type 2<br>endothelial cells, eosinophils/basophils, fibroblasts/pericytes, chondrocytes, or NK cells due to ongoing development<br>of antibody-oligonucleotide conjugates with sufficient specificity for these markers. Protein targets were detected<br>using barcode-conjugated antibodies either sourced directly from Akoya Biosciences or produced via custom conjugation<br>using Akoya’s antibody conjugation kit (Cat# 232195). Tissue sections underwent antigen retrieval in AR9 Buffer<br>(Akoya, PN#AR9001KT) using a pressure cooker for 20 minutes, along with autofluorescence quenching. For optimal<br>imaging of proximal lung tissues containing large airway and vascular features, antibodies targeting extracellular<br>matrix, endothelium, and vascular smooth muscle should be imaged within the first five cycles to minimize structural<br>degradation. Pan markers (e.g., PanCK, ACTA2) can be reserved for later cycles. Image analysis was performed using a<br>combination of segmentation tools, vascular and airway feature annotation, and export of masks and annotations, as<br>described in (Purkerson, Pryhuber, Colon, et al. 2025b). The antibody validation protocol is available in a separate<br>reference (Purkerson, Pryhuber, Huyck, et al. 2025).<br><br>**Bibliography:**<br><br>* Pryhuber, Gloria. 2021. “Lung (v1.2) Graph Data, v1.2.” https://lod.humanatlas.io/asct-b/lung/v1.2/.<br>* Purkerson, Jeffrey, Gloria S. Pryhuber, Luis Colon, and Heidie Huyck. 2025a. “813.1 Multiplexed Immunofluorescence Phenocycler-Fusion® Imaging of FFPE Lung Sections.” https://www.protocols.io/view/813-1-multiplexed-immunofluorescence-phenocycler-f-dt3j6qkn.<br>* Purkerson, Jeffrey, Gloria S. Pryhuber, Luis Colon, and Heidie Huyck. 2025b. “813.1 Multiplexed Immunofluorescence Phenocycler-Fusion® Imaging of FFPE Lung Sections.” https://www.protocols.io/view/813-1-multiplexed-immunofluorescence-phenocycler-f-dt3j6qkn.<br>* Purkerson, Jeffrey, Gloria S. Pryhuber, Heidie Huyck, and Gail Deutsch. 2025. “812.2 Lung FFPE Multiplexed Immunofluorescence Phenocycler-Fusion® Antibody Validation Protocol.” https://www.protocols.io/view/812-2-lung-ffpe-multiplexed-immunofluorescence-phe-dqjn5ume.