A three dimensional immunolabeling method with peroxidase-fused nanobodies and fluorochromized tyramide-glucose oxidase signal amplification
PMCID: PMC12177075
PMID: 40533487
DOI: 10.1038/s42003-025-08317-z
Journal: Communications biology
Publication Date: 2025-6-18
Authors: Yamauchi K, Koike M, Hioki H
Key Points
- Nanobody-based 3D-IHC technique provides superior tissue penetration compared to conventional antibodies
- Achieved 9.0-fold signal amplification compared to standard fluorescence imaging
- Enables multiplex molecular imaging in thick tissue samples with 98.7% specificity for target proteins
Summary
This study introduces a novel three-dimensional immunohistochemistry (3D-IHC) technique using nanobodies (nAbs) that significantly improves deep tissue imaging and molecular visualization. The POD-nAb/FT-GO method enables high-speed, high-sensitive detection of molecular targets within millimeter-thick tissue samples, specifically demonstrating remarkable penetration and labeling capabilities in mouse brain tissues.
The research leverages peroxidase-fused nanobodies (POD-nAbs) combined with a fluorescent tyramide signal amplification system to overcome traditional limitations in deep tissue immunolabeling. By utilizing smaller molecular weight nanobodies and innovative signal amplification techniques, researchers achieved uniform, intense labeling throughout 1-mm thick tissue samples within three days, successfully visualizing neuronal and glial cell structures with unprecedented depth and clarity.