OMAP-18: Organ Mapping Antibody Panel (OMAP) for Multiplexed Antibody-Based Imaging of human lymph node with Cell DIVE-IBEX, v1.0

OMAP-18 was designed for Cell DIVE imaging (https://doi.org/10.1073/pnas.1300136110) of human FFPE lymph node samples<br>using the IBEX (Iterative Bleaching Extends multi-pleXity) dye inactivation protocol. The resulting method, termed<br>Cell DIVE-IBEX, allows complete signal elimination without tissue loss using IBEX compatible fluorophores<br>(https://doi.org/10.1038/s41596-021-00644-9). The panel consists of 3 cycles of 11 primary antibodies, 4 secondary<br>antibodies, a streptavidin conjugate to detect a biotinylated KI67 antibody (Thermo Fisher Scientific S21384), and<br>nuclear label Hoechst for image alignment and nuclear segmentation. This OMAP provides a spatial context for 15<br>anatomical structures and 11 cell types present in the ASCT+B lymph node table, v1.1<br>(https://doi.org/10.48539/HBM573.SHCQ.259). OMAP-18 was designed using OMAP-1<br>(https://doi.org/10.48539/HBM458.NSWR.957) as a reference and therefore contains all the biomarkers included in<br>OMAP-1. However, OMAP-18 is limited to essential markers for evaluating changes in lymph node architecture and immune<br>cell composition arising from malignancy based on discovery studies using OMAP-1. For this reason, OMAP-18 does not<br>allow granular phenotyping of fibroblastic reticular cells, T regulatory cells, T helper cells, blood and lymphatic<br>endothelial cells, and dendritic cells but can easily be expanded in the future. Importantly, this panel utilizes the<br>BCL2 clone preferred by pathologists (clone SP66), providing additional value for translational studies. Human FFPE<br>samples were prepared using a dual antigen retrieval approach consisting of 30 minutes with Epitope Retrieval Solution<br>1 (pH 6, 100C) and then 30 minutes with Epitope Retrieval Solution 2 (pH 9, 100C) performed on the Leica Bond. More<br>details on the antigen retrieval protocol and step-by-step application of antibodies can be found on the IBEX Imaging<br>Community site (https://doi.org/10.5281/zenodo.7693278). Detailed B cell follicle and cellular community analysis was<br>performed as described in the accompanying manuscript (https://doi.org/10.1016/j.ccell.2024.02.001). Representative<br>datasets can be accessed on Image Data Resource (https://idr.openmicroscopy.org) under accession number idr0158 and<br>querying images with the prefix “CellDIVE_IBEX_Immune.”