Protocol for optical, aqueous-based clearing of murine tissues using EZ Clear

Taeyong Ahn; Gabrielle E Largoza; Julia Younis; RE-JOIN Consortium; Mary E Dickinson; Chih-Wei Hsu; Joshua D Wythe

doi:10.1016/j.xpro.2024.103053

Protocol for optical, aqueous-based clearing of murine tissues using EZ Clear

STAR Protoc. 2024 May 4;5(2):103053. doi: 10.1016/j.xpro.2024.103053. Online ahead of print.

Authors

Taeyong Ahn¹, Gabrielle E Largoza¹, Julia Younis²; RE-JOIN Consortium; Mary E Dickinson³, Chih-Wei Hsu⁴, Joshua D Wythe⁵

Affiliations

¹ Department of Cell Biology, University of Virginia School of Medicine, Charlottesville, VA 22903, USA.
² Department of Human and Molecular Genetics, Baylor College of Medicine, Houston, TX 77030, USA.
³ Department of Integrative Physiology, Baylor College of Medicine, Houston, TX 77030, USA.
⁴ Department of Integrative Physiology, Baylor College of Medicine, Houston, TX 77030, USA; Optical Imaging and Vital Microscopy Core, Advanced Technology Cores, Baylor College of Medicine, Houston, TX 77030, USA; Department of Education, Innovation and Technology, Baylor College of Medicine, Houston, TX 77030, USA. Electronic address: loganh@bcm.edu.
⁵ Department of Cell Biology, University of Virginia School of Medicine, Charlottesville, VA 22903, USA; Department of Neuroscience, University of Virginia School of Medicine, Charlottesville, VA 22903, USA; Brain, Immunology and Glia (BIG) Center, University of Virginia School of Medicine, Charlottesville, VA 22903, USA; UVA Comprehensive Cancer Center, University of Virginia School of Medicine, Charlottesville, VA 22903, USA; Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA 22903, USA. Electronic address: jwythe@virginia.edu.

Abstract

Tissue clearing is an essential prerequisite for 3D volumetric imaging of larger tissues or organs. Here, we present a detailed protocol for optical, aqueous-based clearing of adult murine tissues using EZ Clear. We describe steps to ensure successful perfusion and fixation of organs from the adult mouse and supply guidelines for optimal lipid removal, refractive index matching, and tissue clearing. Finally, we provide imaging parameters for visualizing both exogenous perfused fluorescent dyes and endogenous fluorescence reporters in the adult mouse. For complete details on the use and execution of this protocol, please refer to Hsu et al.¹.

Keywords: Developmental biology; Model Organisms; Neuroscience; microscopy.