OMAP-24: Organ Mapping Antibody Panel (OMAP) for Multiplexed Antibody-Based Imaging of Human Pancreas with CyCIF, v1.0

OMAP-24 was developed using cyclic immunofluorescence (CyCIF) to detect 13 protein biomarkers in the human pancreas,<br>with a specific emphasis on pancreatic ganglia and nervous innervation. Human FFPE pancreas samples were fixed in 4%<br>paraformaldehyde (PFA) and processed according to the Human Pancreas Processing protocol (Chen et al. 2022). This<br>panel contributes spatial data for anatomical structures and cell types defined in the ASCT+B pancreas table v1.3<br>(Campbell-Thompson, Eskaros, and Saunders 2023), with a focus on nervous structures, an area not previously addressed<br>in earlier OMAPs. Images from sequential CyCIF staining rounds were aligned using nuclear markers DAPI (cycle 1) and<br>Hoechst S769121 (cycles 2 and 3). Antigen retrieval was performed at 95°C for 25 minutes in 1X AR9 solution using a<br>vegetable steamer. Detection involved a combination of OPAL TSA fluorophores and Alexa Fluor-conjugated secondary<br>antibodies. Primary antibodies requiring TSA amplification used various HRP polymer reagents from Vector Laboratories<br>and Akoya Biosciences. No antibody cycle sensitivity was observed during testing. OMAP-24 builds upon earlier<br>pancreas-focused panels (OMAP-6 and OMAP-13), but uniquely adds spatial mapping of pancreatic ganglia and nerve<br>innervation. This is the first OMAP to focus on the neuroanatomy of the pancreas, providing insights relevant to<br>pancreatic disorders such as type 1 diabetes. Glial, interstitial, and immune cells were not included in this version<br>due to limitations in antibody availability. The full staining protocol is available at Ewing et al. (2025), and a<br>sample dataset can be accessed at Zenodo (Ewing and Campbell-Thompson 2025).<br><br>**Bibliography:**<br><br>* Campbell-Thompson, Martha, Adel Eskaros, and Diane Saunders. 2023. “Pancreas (v1.3) Graph Data, v1.3.” https://lod.humanatlas.io/asct-b/pancreas/v1.3/.<br>* Chen, Jing, Edward J. Butfiloski, cxm, and Martha Campbell-Thompson. 2022. “Human Pancreas Processing.” https://www.protocols.io/view/human-pancreas-processing-b7gxrjxn.<br>* Ewing, Samuel, and Martha Campbell-Thompson. 2025. “Pancreas Organ Mapping Antibody Panel (OMAP) Using CyCIF to Stain Pancreatic Ganglia.” Zenodo. https://zenodo.org/records/14867745.<br>* Ewing, Samuel, Hong Huang, Sydney Haselden, Lith Nasif, and Martha Campbell-Thompson. 2025. “Multiplex Immunofluorescence for Human Pancreatic Ganglia.” https://doi.org/10.17504/protocols.io.261ger4djl47/v1.<br>* Martinez Casals, Anna, and Frida Björklund. 2024. “6-Pancreas-Codex (v1.3) Graph Data, v1.3.” https://lod.humanatlas.io/omap/6-pancreas-codex/v1.3/.<br>* Saunders, Diane, and Conrad Reihsmann. 2024. “13-Pancreas-Codex (v1.1) Graph Data, v1.1.” https://lod.humanatlas.io/omap/13-pancreas-codex/v1.1/.