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

抽象的。分子表型导致越来越多的赞赏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。除了神经胶质细胞或神经元细胞的一级分类之外， 兴奋性或抑制性，现在认识到，有几十种分子定义的细胞类型， 它们的形态、连接、生理以及基因和蛋白质表达。固定组织中蛋白质的检测 通过免疫组织化学（IHC）的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，接近脉管系统和细胞外沉积物）提供必要的 生理背景这些数据已经产生了丰富的图片，细胞地形如何因大脑区域而异 并提供了一个强大的背景，以评估细胞类型特异性变化发生在疾病状态，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的流行，其 由于诸如IgG抗体的试剂被动地以缓慢的速率进行反应， 扩散到组织中。由于这一瓶颈，组织传统上被切成薄片（≤ 50 µm），以便于 特征的均匀染色并使定量分析可靠。iDISCO和相关技术 通过去除细胞膜脂质使完整组织光学透明， IHC，因为脱脂赠款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的 瓶颈，完整的样品需要数周至数月的孵育才能到达中心。如果完整 器官可以更快地标记，实际上它将提供一个强大的工具来执行公正的 哺乳动物发育和神经系统疾病模型的分子表型分析。为此，LifeCanvas 开发了SmartLabel（SL），这是世界上第一个全器官主动免疫染色设备， 小鼠大脑在短短24小时内使用专有的随机电转运技术。SL还采用了 亲和力梯度，抗体在结合靶点之前均匀分布在整个组织中的方法 蛋白质产生从样品表面到其核心强度惊人均匀的标记。期间 第一阶段，我们通过（1）扩展SL的组织快速免疫标记能力， 使用iDISCO进行处理，（2）确保与关键形态学、细胞类型和神经元活性的相容性 标记物如c-Fos，（3）使该技术适用于不同的样品类型，如人脑 类器官，以及（4）开发下一代SL原型，同时具有成本效益 多个类器官或成年小鼠脑的群水平免疫标记。在完成了第一阶段的所有工作后， 项目目标，我们现在准备-在第二阶段-完成下一代SL的开发，一个双 一种能够快速免疫染色和交钥匙批量处理的功能清除和标记装置 一系列神经样品类型，包括不同CNS/PNS来源和各种哺乳动物物种的组织。 此外，我们将继续进行内部研究，以扩大经验证的抗 神经科学相关的目标增加了三倍，并进一步使DISCO用户能够在其管道中采用SL， 和均匀的免疫标记。我们的第二阶段目标是商业化的下一代SL，普及积极的 免疫标记并由此促进定量全样品分子表型分析应用 更广泛的神经科学社区。

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.