Yamauchi Kenta, Koike Masato, Hioki Hiroyuki
Department of Neuroanatomy, Juntendo University Graduate School of Medicine, Tokyo, Japan.
Department of Cell Biology and Neuroscience, Juntendo University Graduate School of Medicine, Tokyo, Japan.
Commun Biol. 2025 Jun 18;8(1):903. doi: 10.1038/s42003-025-08317-z.
Three dimensional immunohistochemistry (3D-IHC), immunolabeling of 3D tissues, reveals the spatial organization of molecular and cellular assemblies in the context of the tissue architecture. Deep and rapid penetration of antibodies into 3D tissues and highly sensitive detection are critical for high-throughput imaging analysis of immunolabeled 3D tissues. Here, we report a nanobody (nAb)-based 3D-IHC, POD-nAb/FT-GO 3D-IHC, for high-speed and high-sensitive detection of targets within 3D tissues. Peroxidase-fused nAbs (POD-nAbs) enhanced immunolabeling depth and allowed for highly sensitive detection by combined with a fluorescent tyramide signal amplification system, Fluorochromized Tyramide-Glucose Oxidase (FT-GO). Multiplex labeling was implemented to the 3D-IHC by quenching POD with sodium azide. Using the 3D-IHC technique, we successfully visualized somata and processes of neuronal and glial cells in millimeter-thick mouse brain tissues within three days. Given its high-speed and high-sensitive detection, our 3D-IHC protocol, POD-nAb/FT-GO 3D-IHC, would provide a useful platform for histochemical analysis in 3D tissues.
三维免疫组织化学（3D-IHC），即对三维组织进行免疫标记，可揭示组织结构背景下分子和细胞集合体的空间组织。抗体深入快速渗透到三维组织中以及高灵敏度检测对于免疫标记三维组织的高通量成像分析至关重要。在此，我们报告一种基于纳米抗体（nAb）的3D-IHC，即POD-nAb/FT-GO 3D-IHC，用于高速、高灵敏度检测三维组织内的靶标。过氧化物酶融合纳米抗体（POD-nAbs）增强了免疫标记深度，并通过与荧光酪胺信号放大系统荧光素化酪胺-葡萄糖氧化酶（FT-GO）结合实现高灵敏度检测。通过用叠氮化钠淬灭POD对3D-IHC进行多重标记。使用3D-IHC技术，我们在三天内成功可视化了毫米厚小鼠脑组织中神经元和神经胶质细胞的胞体和突起。鉴于其高速和高灵敏度检测，我们的3D-IHC方案POD-nAb/FT-GO 3D-IHC将为三维组织的组织化学分析提供一个有用的平台。
