OMAP-17: Organ Mapping Antibody Panel (OMAP) for Multiplexed Antibody-Based Imaging of Thymus with IBEX, v1.1

OMAP-17 was developed for IBEX (Iterative Bleaching Extends multi-pleXity) imaging of fixed frozen human pediatric<br>thymus tissue samples (Radtke et al. 2022; 2020). The panel spans 9 imaging cycles and includes 43 primary antibodies,<br>6 secondary antibodies, and Hoechst nuclear dye for image alignment and segmentation. It provides spatial context for<br>7 anatomical structures and over 30 cell types and developmental states (e.g., dividing, immature, precommitted) from<br>the ASCT+B thymus table v1.4 (Brusko, Beuschel, and Radtke 2023). This is the first OMAP developed specifically for<br>the thymus and was designed to detect rare thymic epithelial cell (TEC) populations such as myo-TECs and neuro-TECs,<br>informed by scRNA-seq and CITE-seq studies. OMAP-17 shares markers with other lymphoid tissue OMAPs, including OMAP-1<br>(lymph node), OMAP-10 (tonsil), and OMAP-11 (spleen). However, it does not include markers for plasma cells or<br>ionocyte/tuft TECs. CD1a, a key thymocyte marker, was omitted due to steric hindrance during multi-cycle labeling.<br>Some antibodies were custom conjugated in-house or by suppliers (e.g., Alexa Fluor 532, iFluor 594). Three antibodies<br>used in this OMAP have since been discontinued: KRT10 (NBP2-54402PE), LY75 (NBP2-75467), and Z25303 from Thermo<br>Fisher. This panel contributed to the Human Cell Atlas spatial thymus atlas project. Imaging data can be explored on<br>the Image Data Resource (https://idr.openmicroscopy.org, project-3051), and detailed protocols and examples are<br>available on the IBEX Knowledge-Base (Radtke et al. 2025; Yaniv et al. 2025). Further information on image analysis,<br>integration, and the thymus coordinate framework is described in Yayon et al. (2024).<br><br>**Bibliography:**<br><br>* Brusko, Maigan, Rebecca T. Beuschel, and Andrea J. Radtke. 2023. “Thymus (v1.4) Graph Data, v1.4.” https://lod.humanatlas.io/asct-b/thymus/v1.4/.<br>* Image Data Resource. 2025. “Webclient for Image Data Resource.” https://idr.openmicroscopy.org/webclient/?show=project-3051.<br>* Müller, Werner, Silvia Rüberg, and Andreas Bosio. 2023. “10-Palatine-Tonsil-Mics (v1.0) Graph Data, v1.0.” https://lod.humanatlas.io/omap/10-palatine-tonsil-mics/v1.0/.<br>* Radtke, Andrea J. 2023a. “1-Human-Lymph-Node-Ibex (v1.3) Graph Data, v1.3.” https://lod.humanatlas.io/omap/1-human-lymph-node-ibex/v1.3/.<br>* Radtke, Andrea J. 2023b. “11-Spleen-Ibex (v1.0) Graph Data, v1.0.” https://lod.humanatlas.io/omap/11-spleen-ibex/v1.0/.<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>* Yayon, Nadav, et al. 2024. “A Spatial Human Thymus Cell Atlas Mapped to a Continuous Tissue Axis.” *Nature* 635 (8039): 708–18. https://doi.org/10.1038/s41586-024-07944-6.<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.