3D molecular phenotyping of intact brain tissue via high-throughput active immunohistochemistry

抽象的。分子表型分析使人们越来越了解神经细胞类型的多样性和功能 从而改变了我们对大脑的理解。除了将细胞一级分类为神经胶质细胞或神经元细胞外， 兴奋性或抑制性，现在已经认识到有几十种不同的分子定义的细胞类型 它们的形态、连通性、生理学以及基因和蛋白质的表达。固定组织中蛋白质的检测 免疫组织化学(IHC)一直是细胞类型发现的主要驱动力，细胞的精确 组织内的微环境(例如，靠近血管和细胞外沉积)提供必要的 生理背景。这些数据提供了一幅丰富的图像，展示了细胞的形态如何随大脑区域的变化而变化 并提供了一个强大的背景来评估在疾病状态下发生的特定于细胞类型的变化，例如 严重的神经系统疾病，如阿尔茨海默氏症和帕金森氏症。尽管IHC盛行，但其 由于免疫球蛋白抗体等试剂被动使用的速度太慢，应用一直受到阻碍 扩散到组织中。由于这一瓶颈，组织传统上被薄切(≤50微米)以便于 统一的特征染色，并使定量分析可靠。Clarity、iDISCO和相关技术 通过去除细胞膜脂质来光学清除完整的组织提供了一种执行全脑 IHC，因为脱脂使试剂更容易进入深层组织部位。然而，标签时间仍然是一个主要因素 瓶颈，完整的样品需要几周到几个月的孵化时间才能贴上标签到达中心。如果是完整的 器官可以更快地被标记，实际上它将提供一个强大的工具来公正地执行 哺乳动物发育和神经疾病模型中的分子表型。为此，生活画布 开发了SmartLabel(SL)，这是世界上第一个完整的器官主动免疫染色设备，可以完全标记整个 使用专有的随机电传输技术，小鼠的大脑只需24小时就能完成。SL还使用了一个 亲和力梯度，一种抗体在与靶结合之前均匀分布在组织中的方法 蛋白质产生从样本表面到其核心的强度惊人地均匀的标记。在.期间 第一阶段，我们通过(1)扩展SL对组织的快速免疫标记能力来扩大SL的应用 使用IDISCO处理，(2)确保与关键形态、细胞类型和神经元活动兼容 标记物，如c-Fos，(3)使技术适用于不同类型的样本，如人脑 有机化合物，以及(4)开发同时具有成本效益的下一代SL原型 多种有机化合物或成年小鼠大脑的队列水平免疫标记。已完成所有第一阶段 项目目标，我们现在准备-在第二阶段-完成下一代SL的开发，一对 一种可快速免疫染色、交钥匙批量处理的功能清标装置 一系列神经样本类型，包括不同CNS/PNS来源的组织和各种哺乳动物物种的组织。 此外，我们将继续内部研究，以扩大有效的抗体数量。 神经科学相关目标增加三倍，并进一步使迪斯科用户能够在他们的管道中快速采用SL 和统一的免疫标记。我们的第二阶段目标是将下一代SL商业化，普及Active 免疫标记，从而促进了定量全样本分子表型分析的应用 更广泛的神经科学界。

Abstract. Molecular phenotyping has led to a growing appreciation of neural cell type diversity and function thereby transforming our understanding of the brain. Beyond first-order classification of cells as glial or neuronal, excitatory or inhibitory, it is now recognized that there are dozens of molecularly-defined cell types that differ in their morphology, connectivity, physiology, and gene & protein expression. Detection of protein in fixed tissues via immunohistochemistry (IHC) has been a major driver of cell type discovery, with a cell’s precise microenvironment within tissue (e.g. proximity to vasculature and extracellular deposits) providing essential physiological context. Such data have yielded a rich picture of how cellular topography varies by brain region and provides a robust backdrop to assess cell type-specific changes that occur in disease states, such as profound neurological disorders like Alzheimer’s and Parkinson’s disease. Despite the prevalence of IHC, its application has remained encumbered by the slow rate at which reagents such as IgG antibodies passively diffuse into tissue. Due to this bottleneck, tissues have traditionally been thinly sliced (≤ 50 µm) to facilitate uniform staining of features and make quantitative analyses reliable. CLARITY, iDISCO, and related techniques that optically-clear intact tissues by removing cell membrane lipids have offered a means to perform whole-brain IHC, as delipidation grants reagents easier access to deep tissue sites. However, labeling time remains a major bottleneck, with intact samples requiring weeks to months of incubation for labeling to reach the center. If whole organs could be labeled more quickly and practically it would provide a powerful tool to perform unbiased molecular phenotyping in mammalian models of development and neurological disorders. To this end LifeCanvas developed SmartLabel (SL), the world’s first whole-organ active immunostaining device that fully labels an entire mouse brain in just 24 hrs using proprietary stochastic electrotransport technology. SL additionally employs an affinity ramp, a method in which antibodies are evenly distributed throughout the tissue before binding to target proteins to produce labeling that is strikingly uniform in intensity from the sample’s surface to its core. During Phase I, we broadened SL’s applications by (1) extending SL’s rapid immunolabeling capability for tissues processed using iDISCO, (2) ensuring compatibility with key morphological, cell type, and neuronal activity markers such as c-Fos, (3) adapting the technology to work with diverse sample types such as human cerebral organoids, and (4) developing a prototype next-generation SL that performed simultaneous and cost-effective cohort-level immunolabeling of multiple organoids or adult mouse brains. Having completed all the Phase I project goals, we are now poised – in Phase II – to complete the development of the next generation SL, a dual function clearing and labeling device capable of rapid immunostaining and turnkey batch-processing of a wide range of neural sample types including tissues of different CNS/PNS origins and of various mammalian species. Additionally, we will continue in-house research to expand the number of validated antibodies against neuroscience-related targets by three-fold, and further enable DISCO users to adopt SL in their pipeline for rapid and uniform immunolabeling. Our Phase II goals are to commercialize the next generation SL, popularize active immunolabeling and thereby facilitate application of quantitative whole-sample molecular phenotyping analyses for a broader neuroscience community.