OMAP-15 Organ Mapping Antibody Panel (OMAP) for Multiplexed Antibody-Based Imaging of Human Intestines with CODEX, v1.1

OMAP-15 was designed for CODEX (CO-Detection by indEXing) imaging of formalin-fixed paraffin-embedded (FFPE) human<br>intestine samples using the Akoya Biosciences PhenoCycler system (Black et al. 2021; Kennedy‐Darling et al. 2021). The<br>panel consists of 54 antibodies and DAPI for nuclear segmentation. This OMAP provides spatial context for anatomical<br>structures and cell types listed in ASCT+B Large Intestine v1.3 (Longacre, Hickey, and Lin 2022) and Small Intestine<br>v1.1 (Longacre 2022). It builds upon OMAP-2 (Radtke et al. 2023) by adapting marker panels from frozen to FFPE tissue.<br>Nineteen markers from OMAP-2 were excluded, and 22 new markers were included: ATP1A1, CD206, COL1A1, CTNNB1, FN1,<br>FOXP3, GP2, HLA-DRA, IGHA1, IGHM, ITGAM, KRT4/5/6a/6b/6c/8/10/13/18, MS4A1, MUC6, PDGFRA/B, POSTN, RORC, SIGLEC8,<br>SPIB, TNC, TPSAB1, and UCHL1. Markers for tuft cells and naive intestinal stem cells (LGR5+, ASCL2+) are not included.<br>Carrier-free antibodies were used whenever possible; if carrier-containing antibodies were standard, custom<br>carrier-free alternatives were ordered. All antibodies were oligo-conjugated using protocols detailed in Wei and Bi<br>(2024a). Protocols for sample preparation and antigen retrieval are described in Wei and Bi (2023), and a<br>representative dataset for this OMAP is available (Wei and Bi 2024b).<br><br>**Bibliography:**<br><br>* Black, Sarah, Darci Phillips, John W. Hickey, Julia Kennedy-Darling, Vishal G. Venkataraaman, Nikolay Samusik, Yury Goltsev, Christian M. Schürch, and Garry P. Nolan. 2021. “CODEX Multiplexed Tissue Imaging with DNA-Conjugated Antibodies.” *Nature Protocols* 16 (8): 3802–35. https://doi.org/10.1038/s41596-021-00556-8.<br>* Kennedy‐Darling, Julia, Salil S. Bhate, John W. Hickey, Sarah Black, Graham L. Barlow, Gustavo Vazquez, Vishal G. Venkataraaman, et al. 2021. “Highly Multiplexed Tissue Imaging Using Repeated Oligonucleotide Exchange Reaction.” *European Journal of Immunology* 51 (5): 1262–77. https://doi.org/10.1002/eji.202048891.<br>* Longacre, Teri. 2022. “Small-Intestine (v1.1) Graph Data, v1.1.” 2022. https://lod.humanatlas.io/asct-b/small-intestine/v1.1/.<br>* Longacre, Teri, John Hickey, and Yiing Lin. 2022. “Large-Intestine (v1.3) Graph Data, v1.3.” 2022. https://lod.humanatlas.io/asct-b/large-intestine/v1.3/.<br>* Radtke, Andrea J., Katy Börner, Neil Kelleher, and Ronald N. Germain. 2023. “2-Intestine-Codex (v1.3) Graph Data, v1.3.” 2023. https://lod.humanatlas.io/omap/2-intestine-codex/v1.3/.<br>* Wei, Bei, and Joanna Bi. 2023. “CODEX FFPE Staining and Fixation,” December. https://www.protocols.io/view/codex-ffpe-staining-and-fixation-c6atzaen.<br>* Wei, Bei, and Joanna Bi. 2024a. “CODEX Antibody Conjugation Protocol,” April. https://www.protocols.io/view/codex-antibody-conjugation-protocol-db2j2qcn.<br>* Wei, Bei, and Joanna Bi. 2024b. “OMAP-15 Human Intestine with CODEX.” Zenodo. https://zenodo.org/records/10963121.

OMAP-15 专为使用Akoya Biosciences PhenoCycler系统（Black等人，2021；Kennedy‐Darling等人，2021）对福尔马林固定石蜡包埋（Formalin-Fixed Paraffin-Embedded，FFPE）人体肠道样本进行CODEX（CO-Detection by indEXing）成像而设计。该标记组合包含54种抗体及用于细胞核分割的DAPI。本OMAP可为ASCT+B大肠版本v1.3（Longacre、Hickey与Lin，2022）及小肠版本v1.1（Longacre，2022）中列出的解剖结构与细胞类型提供空间背景信息。本数据集基于OMAP-2（Radtke等人，2023）构建，将标记组合从冰冻组织适配至FFPE组织。研究人员剔除了OMAP-2中的19个标记物，新增22个标记物：ATP1A1、CD206、COL1A1、CTNNB1、FN1、FOXP3、GP2、HLA-DRA、IGHA1、IGHM、ITGAM、KRT4/5/6a/6b/6c/8/10/13/18、MS4A1、MUC6、PDGFRA/B、POSTN、RORC、SIGLEC8、SPIB、TNC、TPSAB1及UCHL1。本数据集未包含簇细胞与初始肠道干细胞（LGR5+、ASCL2+）的标记物。

研究团队尽可能使用无载体抗体；若含载体抗体为标准试剂，则定制无载体替代抗体。所有抗体均采用寡核苷酸偶联，具体操作流程详见Wei与Bi（2024a）发表的方案。样本制备与抗原修复的操作流程详见Wei与Bi（2023），本OMAP的代表性数据集可于Wei与Bi（2024b）获取。

**参考文献：**

* 莎拉·布莱克、达西·菲利普斯、约翰·W·希基、朱莉娅·肯尼迪-达林、维沙尔·G·文卡特拉曼、尼古拉·萨穆西克、尤里·戈尔捷耶夫、克里斯蒂安·M·许尔希、加里·P·诺兰. 2021. 《使用DNA偶联抗体的CODEX多重组织成像》. *Nature Protocols* 16(8): 3802–35. https://doi.org/10.1038/s41596-021-00556-8.
* 朱莉娅·肯尼迪-达林、萨利尔·S·巴特、约翰·W·希基、莎拉·布莱克、格雷厄姆·L·巴洛、古斯塔沃·巴斯克斯、维沙尔·G·文卡特拉曼等. 2021. 《基于重复寡核苷酸交换反应的超高多重组织成像》. *European Journal of Immunology* 51(5): 1262–77. https://doi.org/10.1002/eji.202048891.
* 特里·朗克雷. 2022. 《小肠（v1.1）图形数据，v1.1》. 2022. https://lod.humanatlas.io/asct-b/small-intestine/v1.1/.
* 特里·朗克雷、约翰·希基、林应. 2022. 《大肠（v1.3）图形数据，v1.3》. 2022. https://lod.humanatlas.io/asct-b/large-intestine/v1.3/.
* 安德里亚·J·拉德克、凯蒂·博尔纳、尼尔·凯勒赫、罗纳德·N·热曼. 2023. 《2-肠道-CODEX（v1.3）图形数据，v1.3》. 2023. https://lod.humanatlas.io/omap/2-intestine-codex/v1.3/.
* 贝·魏、乔安娜·毕. 2023. 《CODEX FFPE染色与固定》，12月. https://www.protocols.io/view/codex-ffpe-staining-and-fixation-c6atzaen.
* 贝·魏、乔安娜·毕. 2024a. 《CODEX抗体偶联操作方案》，4月. https://www.protocols.io/view/codex-antibody-conjugation-protocol-db2j2qcn.
* 贝·魏、乔安娜·毕. 2024b. 《带CODEX的OMAP-15人体肠道数据集》. Zenodo. https://zenodo.org/records/10963121.