OMAP-18: Organ Mapping Antibody Panel (OMAP) for Multiplexed Antibody-Based Imaging of human lymph node with Cell DIVE-IBEX, v1.1

OMAP-18 was developed for Cell DIVE imaging of human FFPE lymph node tissue using the IBEX dye inactivation protocol<br>(Gerdes et al. 2013; Radtke et al. 2020; 2022). This combined approach, termed Cell DIVE-IBEX, uses fluorophores<br>compatible with IBEX to enable complete signal elimination between cycles without tissue loss. The panel includes 3<br>imaging cycles with 11 primary antibodies, 4 secondary antibodies, a streptavidin conjugate for detecting biotinylated<br>KI67 (Thermo Fisher S21384), and the nuclear stain Hoechst for image alignment and segmentation. OMAP-18 maps 15<br>anatomical structures and 11 cell types found in the ASCT+B Lymph Node v1.1 table (Jorgensen, Radtke, and Rodriguez<br>2021). It is a focused version of OMAP-1 (Radtke 2023), retaining key markers to detect malignancy-driven changes in<br>lymph node architecture and immune composition. It excludes markers needed for deep phenotyping of T regulatory/helper<br>cells, fibroblastic reticular cells, dendritic cells, and endothelial subtypes but is expandable. The panel includes<br>the clinically preferred BCL2 antibody clone (SP66), enhancing its utility for translational studies. Antigen<br>retrieval was performed on a Leica Bond system using a dual-step protocol: Epitope Retrieval Solution 1 (pH 6) for 30<br>minutes followed by Epitope Retrieval Solution 2 (pH 9) for 30 minutes at 100°C. Complete protocol details, antibody<br>application steps, and analysis workflows can be accessed via the IBEX Imaging Community (Yaniv et al. 2025). B cell<br>follicle and immune community analyses are described in the companion paper (Radtke et al. 2024). Representative<br>datasets are available at the Image Data Resource (https://idr.openmicroscopy.org, project-3001). Further<br>documentation, training, and community resources are hosted in the IBEX Knowledge-Base (Radtke et al. 2025; Yaniv et<br>al. 2025).<br><br>**Bibliography:**<br><br>* Gerdes, Michael J., et al. 2013. “Highly Multiplexed Single-Cell Analysis of Formalin-Fixed, Paraffin-Embedded Cancer Tissue.” *PNAS* 110 (29): 11982–87. https://doi.org/10.1073/pnas.1300136110.<br>* Image Data Resource. 2025. “Webclient for Image Data Resource.” https://idr.openmicroscopy.org/webclient/?show=project-3001.<br>* Jorgensen, Marda, Andrea J. Radtke, and Natalie Rodriguez. 2021. “Anatomical Structures, Cell Types, plus Biomarkers (ASCT+B) Table for Lymph Node v1.1.” https://doi.org/10.48539/HBM573.SHCQ.259.<br>* Radtke, Andrea J. 2023. “1-Human-Lymph-Node-Ibex (v1.3) Graph Data.” https://lod.humanatlas.io/omap/1-human-lymph-node-ibex/v1.3/.<br>* Radtke, Andrea J., et al. 2022. “IBEX: An Iterative Immunolabeling and Chemical Bleaching Method for High-Content Imaging of Diverse Tissues.” *Nature Protocols* 17 (2): 378–401. https://doi.org/10.1038/s41596-021-00644-9.<br>* Radtke, Andrea J., et al. 2020. “IBEX: A Versatile Multiplex Optical Imaging Approach for Deep Phenotyping and Spatial Analysis of Cells in Complex Tissues.” *PNAS* 117 (52): 33455–65. https://doi.org/10.1073/pnas.2018488117.<br>* Radtke, Andrea J., et al. 2024. “Multi-Omic Profiling of Follicular Lymphoma Reveals Changes in Tissue Architecture and Enhanced Stromal Remodeling in High-Risk Patients.” *Cancer Cell* 42 (3): 444–63.e10. https://doi.org/10.1016/j.ccell.2024.02.001.<br>* Yaniv, Ziv, et al. 2025. “Iterative Bleaching Extends Multiplexity (IBEX) Knowledge-Base.” Zenodo. https://zenodo.org/records/14645972.<br>* Radtke, Andrea J., et al. 2025. “The IBEX Knowledge-Base: A Central Resource for Multiplexed Imaging Techniques.” *PLoS Biol* 23(3): e3003070. https://doi.org/10.1371/journal.pbio.3003070.<br>* Yaniv, Ziv, et al. 2025. “The IBEX Imaging Knowledge-Base: A Community Resource Enabling Adoption and Development of Immunofluorescence Imaging Methods.” *eLife* 14:RP105737.