Rapid sonication-assisted whole tissue clearing and immunostaining

Abstract

Abstract High-resolution mapping of three-dimensional structures in biological tissues is essential for understanding various biological processes. However, the optical heterogeneity of these tissues, marked by varying optical properties, causes light scattering and absorption, complicating imaging. Current tissue clearing methods can take between 48 h and 32 days, and the limited diffusion depth of fluorescent probes restricts whole-tissue imaging. This study introduces an innovative Sonication-Assisted Tissue Clearing and Immunofluorescent Staining method (SoniC/S), which combines low-frequency ultrasound with a commercial chemical clearing kit and iDISCO staining techniques. When tested on the soft tissue of mouse muscle, the dense collagenous tissue of rat tendon, and the heme-rich tissue of mouse spleen, SoniC/S achieved complete clearing in just 36 h and uniform labeling in 15 h. Overall, SoniC/S provides a rapid and effective approach for tissue clearing and deep immunostaining, facilitating high-resolution volumetric imaging in biological research.