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

Area of Science:

• Biological Imaging
• Histology
• Biochemistry

Background:

• 3D volumetric imaging of large tissues requires effective tissue clearing techniques.
• Optical clearing methods are crucial for preserving fluorescence signals in biological samples.
• Existing protocols may have limitations in lipid removal or refractive index matching for specific applications.

Purpose of the Study:

• To present a detailed protocol for aqueous-based optical clearing of adult murine tissues using EZ Clear.
• To provide guidelines for optimizing perfusion, fixation, lipid removal, and refractive index matching.
• To establish imaging parameters for visualizing fluorescent signals in cleared adult mouse tissues.

Main Methods:

• Development and validation of an aqueous-based tissue clearing protocol using EZ Clear.
• Optimization of perfusion and fixation procedures for adult mouse organs.
• Assessment of lipid removal efficiency and refractive index matching.
• Determination of optimal imaging parameters for fluorescence microscopy.

Main Results:

• Successful optical clearing of adult murine tissues using the EZ Clear protocol.
• Optimized steps for perfusion, fixation, lipid removal, and refractive index matching were established.
• Effective visualization of both exogenous fluorescent dyes and endogenous fluorescence reporters was achieved.

Conclusions:

• The presented EZ Clear protocol offers an effective method for aqueous-based optical clearing of adult mouse tissues.
• This protocol facilitates high-resolution 3D volumetric imaging of whole organs.
• The method supports the visualization of various fluorescence signals, advancing biological research.