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Three-Dimensional Imaging of Tumor-Bearing Tissue Using the Iterative Bleaching Extends Multiplexity Approach

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Proposal for a simple and easy-to-implement protocol for three-dimensional tissue imaging that is compatible with

Takuto Matano¹, Kiyotada Naitou¹, Jannatul Ferdous²

¹Department of Basic Veterinary Science, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.

Acta Histochemica

|April 24, 2025

Summary

This study introduces a simplified protocol for three-dimensional (3D) tissue observation using tissue clearing and immunostaining. The new method enhances immunolabeling and is compatible with standard confocal microscopes, promoting wider adoption of 3D tissue imaging.

Keywords:

CUBIC SeeDB2 Serotonin TPH2 Tissue clearing

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Area of Science:

Biotechnology
Microscopy
Histology

Background:

Traditional tissue observation is limited to 2D due to light penetration and antibody access issues.
Emerging 3D tissue observation methods require specialized equipment and complex protocols, hindering widespread use.
Confocal microscopes are widely available, making them ideal for accessible 3D tissue imaging.

Purpose of the Study:

To develop a simple, adaptable protocol for 3D tissue observation compatible with confocal microscopy.
To facilitate the transition from 2D to 3D tissue imaging by optimizing tissue clearing and immunostaining.
To promote broader adoption of advanced 3D tissue visualization techniques.

Main Methods:

Investigated the impact of tissue clearing and immunostaining conditions on thin tissue slices.
Evaluated the efficacy of CUBIC-L in enhancing immunolabeling.
Optimized detergent concentrations and developed a two-step staining procedure.

Main Results:

CUBIC-L effectively enhanced immunolabeling without reducing antigen immunoreactivity.
High detergent concentrations increased the intensity of immunoreactivity.
A two-step staining protocol was identified as suitable for the proposed method.

Conclusions:

A simple and adaptable protocol for 3D tissue observation using tissue clearing and immunostaining has been established.
The protocol is compatible with widely available confocal microscopes.
This work is expected to accelerate the adoption of 3D tissue observation techniques in research.