OMAP-21: Organ Mapping Antibody Panel (OMAP) for Multiplexed Antibody-Based Imaging of Human Palatine Tonsil with MICS on MACSima 1.5

OMAP-21 was designed for MICS (MACSima imaging cyclic staining) imaging of FFPE human tonsil samples. Tissue fixation and antigen retrieval is described in (Spatial protein and RNA analysis on the same tissue section using MICS technology). The MACSima technology is described in detail in the following publication (MACSima imaging cyclic staining (MICS) technology reveals combinatorial target pairs for CAR T cell treatment of solid tumors). Most, but not all, antibodies in this panel are recombinant antibodies with a mutated human IgG1 constant region. The described mutation removes the Fc receptor binding capacity of human IgG1, eliminating the need for additional blocking steps and reducing non-specific binding of human antibodies on human tissues. Highly multiplexed imaging is achieved through cycles of immunolabeling with FITC, PE, and APC conjugated antibodies and photobleaching to eliminate fluorescence signal between imaging cycles. The panel contains 47 antibodies and the nuclear marker DAPI for image alignment and nuclear segmentation. This OMAP provides a spatial context for all anatomical structures and most cell types present in the human palatine tonsil. OMAP-21 follows closely OMAP-1 described for human lymph nodes (omap-1-human-lymph-node-ibex) and OMAP-10 (omap-10-palatine-tonsil-macsima). All reagents were obtained from Miltenyi Biotec and have been rigorously tested through an internal quality control system to have minimal variation between lots. For this reason, lot information is not included in this table. Analysis was performed by an accompanied software package MACSIQ View Analysis also described in the MACSima publication (https://doi.org/10.1038/s41598-022-05841-4). The result of the analysis is included in the uploaded dataset. In brief, the software processes the raw images of the MACSima run, generates stitched images, and then allows downstream analysis including cell segmentation, cell gating, data normalization, dimension reduction plots (tSNE, UMAP), heat maps, distance analyses and cluster analyses, all of which are interactively linked together. The MACSima system is continuously evolving, this is the second OMAP for the MACSima system. A representative dataset created using OMAP-21 can be found here: 10.5281/zenodo.11281609 .OMAP-21专为福尔马林固定石蜡包埋（Formalin-Fixed Paraffin-Embedded, FFPE）人类扁桃体样本的MACSima循环染色成像（MACSima imaging cyclic staining, MICS）技术设计。组织固定与抗原修复的相关方法已在《基于MICS技术的同一组织切片空间蛋白质与RNA分析》（Spatial protein and RNA analysis on the same tissue section using MICS technology）一文中进行了阐述。MACSima技术的详细信息可参阅《MACSima循环染色成像（MICS）技术揭示嵌合抗原受体T细胞（Chimeric Antigen Receptor T Cell, CAR-T）治疗实体瘤的组合靶点对》（MACSima imaging cyclic staining (MICS) technology reveals combinatorial target pairs for CAR T cell treatment of solid tumors）一文。本抗体组的绝大多数（而非全部）抗体为携带突变的人源IgG1恒定区的重组抗体。该突变可消除人源IgG1的Fc受体结合能力，省去额外的封闭步骤，同时降低人抗体在人体组织上的非特异性结合。通过使用异硫氰酸荧光素（FITC）、藻红蛋白（PE）和别藻蓝蛋白（APC）标记的抗体进行免疫标记循环，并结合光漂白以消除成像循环间的荧光信号，从而实现超高多重成像。本抗体组包含47种抗体，以及用于图像配准和细胞核分割的细胞核标记物4',6-二脒基-2-苯基吲哚（DAPI）。本OMAP可为人腭扁桃体中所有解剖结构与多数细胞类型提供空间分布信息。OMAP-21与此前针对人类淋巴结开发的OMAP-1（标识为omap-1-human-lymph-node-ibex）以及OMAP-10（标识为omap-10-palatine-tonsil-macsima）高度相似。 所有试剂均购自美天旎生物技术（Miltenyi Biotec），并通过内部质量控制系统进行了严格检测，确保不同批次间的差异极小。因此本表格未包含批次信息。数据分析采用配套软件包MACSIQ View Analysis完成，该软件的相关信息已在MACSima相关文献（https://doi.org/10.1038/s41598-022-05841-4）中进行了详细说明。本次分析的结果已包含于上传的数据集中。简言之，该软件可对MACSima成像生成的原始图像进行处理，生成拼接图像，并支持后续的细胞分割、细胞门控、数据归一化、降维可视化（tSNE、UMAP）、热图绘制、距离分析及聚类分析等操作，所有分析模块均可实现交互式联动。MACSima系统仍在持续迭代更新，本数据集是针对该系统开发的第二款OMAP。可通过以下链接获取使用OMAP-21生成的代表性数据集：10.5281/zenodo.11281609。