A NANOBODY-BASED THREE-DIMENSIONAL IMMUNOLABELING METHOD WITH FLUOROCHROMIZED TYRAMIDE-GLUCOSE OXIDASE SIGNAL AMPLIFICATION

Three-dimensional immunohistochemistry (3D-IHC) enables the visualization of molecular and cellular distributions within volumetric tissues, providing insights into the spatial organization of molecules within tissue architecture. Uniform antibody penetration into deep tissues and high-sensitivity detection are essential for high-throughput volumetric imaging. However, conventional immunolabeling techniques exhibit limited penetration depth and often cause signal accumulation at the tissue periphery in thick specimens, hindering reliable quantification and comprehensive structural analysis. Recent advances, including tissue clearing and optimized permeabilization protocols, have improved antibody access; nevertheless, robust and homogeneous deep labeling across millimeter-scale tissues remains insufficient. Nanobodies (nAbs), single-domain antigen-binding fragments derived from camelids, represent promising probes for 3D labeling owing to their small molecular size and high diffusivity compared with conventional immunoglobulins. However, their signal intensity is often insufficient without amplification, limiting their utility in volumetric applications. To address these challenges, we developed a nanobody-based 3D-IHC method that combines peroxidase-fused nanobodies (POD-nAbs) with a fluorochromized tyramide–glucose oxidase (FT-GO) signal amplification system. This approach enables rapid and highly sensitive detection even in thick tissues, such as millimeter-thick mouse brain slices, and overcomes major barriers in antibody penetration and signal detection, providing a versatile platform for detailed morphofunctional analysis of large biological samples.