Detection of Target Molecules Within One-Millimeter-Thick Mouse Brain Slices by Using Peroxidase-Fused Nanobodies and...

Three-dimensional immunohistochemistry (3D-IHC) shows the organization of molecular assemblies in the context of tissue architecture. Deep and rapid antibody penetration into 3D tissues and highly sensitive detection are crucial for high-throughput analysis of 3D-IHC imaging. Here, we provide a detailed protocol for a nanobody (nAb)-based 3D-IHC technique, namely POD-nAb/FT-GO 3D-IHC, for high-speed and high-sensitivity detection of targets within 1-mm-thick mouse brain tissues. Peroxidase-fused nAb (POD-nAb) is a genetically encoded recombinant antibody, which consists of a camelid nAb and a variant of horseradish peroxidase, and fluorochromized tyramide-glucose oxidase (FT-GO) is a fluorescent tyramide signal amplification (TSA) system. POD-nAb/FT-GO 3D-IHC incorporates three main components: 1) tissue permeabilization, 2) POD-nAb binding, and 3) 3D-TSA reaction with FT-GO. POD-nAbs enhance signal penetration depth and allow for highly sensitive detection when combined with FT-GO signal amplification. By using the 3D-IHC protocol provided herein, we can visualize target molecules in mouse brain tissues of 1-mm thickness with drastic signal enhancement within three days. This protocol for POD-nAb/FT-GO 3D-IHC could facilitate structural and molecular interrogation of 3D tissues. Key features • A high-speed 3D-IHC technique that combines POD-nAbs and FT-GO signal amplification, offering faster results than conventional 3D-IHC using IgG antibodies. • Visualization of somata and processes of neuronal and glial cells in millimeter-thick mouse brain tissues within three days. • POD-nAb/FT-GO 3D-IHC offers 9.0- and 6.8-fold signal amplification compared with EGFP fluorescence and 3D-IHC using a synthetic fluorophore-conjugated anti-GFP nAb, respectively. • POD-nAbs, camelid nanobodies fused with peroxidase, are immunoreagents with high specificity, selectivity, and reproducibility.