OMAP-4 Organ Mapping Antibody Panel (OMAP) for Multiplexed Antibody-Based Imaging of Human Skin with Cell DIVE, v1.4

OMAP-4 was designed for Cell DIVE multiplexed imaging of human formalin fixed paraffin embedded (FFPE) skin. Cell DIVE<br>enables highly multiplexed single cell analysis using antibody-based imaging (Gerdes et al. 2013). The tissue slides<br>were prepared with a two-step antigen retrieval process (pH 6 and pH 9) as described here: (McDonough et al. 2020).<br>Antigens were detected using either primary antibodies with fluorophore-labeled secondaries or fluorescent dye<br>conjugated antibodies. The protocol can be found here: (Pryhuber et al. 2022). The panel contains 18 antibodies and<br>the nuclear marker DAPI for image registration and nuclear segmentation. This OMAP provides a spatial context for most<br>anatomical structures and cell types present in the ASCT+B skin table, v1.1 (Ginty, Ho, and Sunshine 2021).<br>Keratinocyte populations can be identified based on their spatial location, distinct morphology and, in the case of<br>corneocytes, absence of nuclei. Additionally, the inclusion of protein biomarkers DDB2 and p53 allow profiling of skin<br>damage and aging effects. This panel covers diverse cell types in the skin and will be further developed to include<br>markers for dermal dendritic cells (DCs) such as Langerin and CD11c. This panel does not include markers targeting<br>several immune cell subsets including Langerhans cells, mast cells, and gamma delta T cells. Representative datasets<br>created using this OMAP are publicly available on the HuBMAP data portal (Quardokus et al. 2023). This revised v1.4<br>OMAP corrects CD3D to CD3E.<br><br>**Bibliography:**<br><br>* Gerdes, Michael J., Christopher J. Sevinsky, Anup Sood, Sudeshna Adak, Musodiq O. Bello, Alexander Bordwell, Ali Can, et al. 2013. “Highly Multiplexed Single-Cell Analysis of Formalin-Fixed, Paraffin-Embedded Cancer Tissue.” *Proceedings of the National Academy of Sciences* 110 (29): 11982–87. https://doi.org/10.1073/pnas.1300136110.<br>* Ginty, Fiona, Jonhan Ho, and Joel Sunshine. 2021. “Skin (v1.1) Graph Data, v1.1.” https://lod.humanatlas.io/asct-b/skin/v1.1/.<br>* McDonough, Liz, Chrystal Chadwick, Fiona Ginty, Christine Surrette, and Anup Sood. 2020. “Cell DIVETM Platform | Slide Clearing and Antigen Retrieval,” December. https://doi.org/10.17504/protocols.io.bpwumpew.<br>* Pryhuber, Gloria, gail.deutsch, Christine Surrette, Lisa Lowery, heidie_huyck, and Cory Poole. 2022. “HuBMAP | GE/URMC/SCH Cell DIVETM Modality Overview,” February. https://doi.org/10.17504/protocols.io.b4kgqutw.<br>* Quardokus, Ellen M., Diane C. Saunders, Elizabeth McDonough, John W. Hickey, Christopher Werlein, Christine Surrette, Presha Rajbhandari, et al. 2023. “Publication Page Organ Mapping Antibody Panels: A Community Resource for Standardized Multiplexed Tissue Imaging.” *HuBMAP Data Portal*. July 19, 2023. https://portal.hubmapconsortium.org/browse/publication/e8338966c69e759157d6c6ad24847989.

OMAP-4专为人类福尔马林固定石蜡包埋（FFPE）皮肤样本的Cell DIVE多重成像实验设计。Cell DIVE可基于抗体成像技术实现超高多重单细胞分析（Gerdes等，2013）。组织玻片采用两步抗原修复工艺（pH 6与pH 9）制备，具体流程参见McDonough等（2020）的文献。抗原检测可采用荧光标记二抗标记一抗，或直接使用荧光染料偶联抗体的方案，详细实验流程参见Pryhuber等（2022）。该抗体组合包含18种抗体以及用于图像配准与细胞核分割的核标记物DAPI。此OMAP可为ASCT+B皮肤数据集v1.1（Ginty、Ho与Sunshine，2021）中收录的绝大多数解剖结构与细胞类型提供空间背景信息。角质形成细胞群可通过其空间位置、独特形态进行鉴定；对于角化细胞（corneocytes）而言，还可通过无细胞核的特征进行区分。此外，该组合包含蛋白质生物标志物DDB2与p53，可用于分析皮肤损伤与衰老相关特征。该抗体组合覆盖皮肤内多种细胞类型，后续将进一步添加真皮树突状细胞（DCs）的标记物，例如Langerin与CD11c。但该组合未包含针对多种免疫细胞亚群的标记物，包括朗格汉斯细胞、肥大细胞以及γδ T细胞。采用此OMAP构建的代表性数据集可在HuBMAP数据门户（Quardokus等，2023）公开获取。此修订版v1.4 OMAP将CD3D修正为CD3E。

**参考文献：**

* Gerdes, Michael J., Sevinsky, Christopher J., Sood, Anup, 等, 2013. "福尔马林固定石蜡包埋癌组织的超高多重单细胞分析". *美国国家科学院院刊*, 110(29): 11982–11987. https://doi.org/10.1073/pnas.1300136110.
* Ginty, Fiona, Ho, Jonhan, Sunshine, Joel, 2021. "皮肤（v1.1）图谱数据v1.1". https://lod.humanatlas.io/asct-b/skin/v1.1/.
* McDonough, Liz, Chadwick, Chrystal, Ginty, Fiona, 等, 2020. "Cell DIVE™平台 | 玻片通透与抗原修复", 2020年12月. https://doi.org/10.17504/protocols.io.bpwumpew.
* Pryhuber, Gloria, Deutsch, Gail, Surrette, Christine, 等, 2022. "HuBMAP | GE/URMC/SCH Cell DIVE™实验方案概述", 2022年2月. https://doi.org/10.17504/protocols.io.b4kgqutw.
* Quardokus, Ellen M., Saunders, Diane C., McDonough, Elizabeth, 等, 2023. "器官图谱抗体组合公开页面：标准化多重组织成像的社区资源". *HuBMAP数据门户*, 2023年7月19日. https://portal.hubmapconsortium.org/browse/publication/e8338966c69e759157d6c6ad24847989.