SOP: Construction of Organ Mapping Antibody Panels for Multiplexed Antibody-Based Imaging of Human Tissues

Optical imaging provides critical spatial data for the vast network of cell types and anatomical structures present in the human body. One such approach, highly multiplexed antibody-based imaging, targets protein biomarkers in situ using dozens of antibodies, allowing for the comprehensive profiling of cell phenotypes at single cell resolution within normal and diseased tissues. Beyond preserving cell-cell interactions and tissue architecture, this approach offers insight into the spatial patterns of complex tissues. This technique also permits assessment of cell states using probes for post-translationally modified molecules generated by active signaling events or by imaging of nuclear localization of transcription factors. When coupled with advanced analytical methods, high content imaging allows for the quantification of rare and difficult to extract cell types such as tissue-resident macrophages and fibroblasts. While these methods may vary in antibody format (fluorophore-, metal-, or oligo-conjugated), mode of imaging (all-in-one or cyclic), or mode of immunolabeling (all-in-one or cyclic), all employ antibodies for protein biomarker detection (see Figure 1b) in (Hickey et al. 2021) . Given the wealth of information provided by multiplexed antibody-based imaging, these techniques are foundational to international efforts aimed at building a comprehensive human reference atlas (HRA) (Snyder et al. 2019; Regev et al. 2017). 

 Spatial mapping approaches pose significant challenges as they are: 1) targeted: antibodies must be carefully validated before data acquisition, 2) fallible: non-reproducible immunolabeling and/or off target labeling is well-described (Uhlen et al. 2016; Bordeaux et al. 2010), 3) resource-intensive: a panel of 50 unique antibodies may require thousands of US dollars in reagent costs and months to build (Hickey et al. 2021), and 4) expertise-dependent:  relying on Subject Matter Experts for their construction and optimization  (Du et al. 2019; Radtke, Chu, et al. 2022; Radtke et al. 2020). To overcome these limitations, we are building Organ Mapping Antibody Panels (OMAPs) that identify the major anatomical structures and cell types present in various human tissues. Furthermore, we are integrating OMAPs with their respective protein biomarkers and cell types in the Anatomical Structures, Cell Types plus Biomarkers (ASCT+B)  tables. To facilitate tissue mapping efforts within and beyond the HuBMAP community, OMAPs are designed for integration with the ASCT+B Reporter  (Börner et al. 2021), a state-of-the-art visualization tool. We strongly encourage inclusion of blood endothelial markers to empower construction of a human reference atlas using the vasculature common coordinate framework (VCCF), originally proposed by (Galis 2020) and expanded upon here (Weber, Ju, and Börner 2020).  The inclusion of antibodies directed against one or more lymphatic endothelial markers (e.g., LYVE1) is highly recommended to further our understanding of the human lymphatic system (Radtke, Lukacs, et al. 2022; National Heart, Lung, and Blood Institute (NHLBI) and National Institutes of Health (NIH) 2021).