Clearing of the Mouse Brain for Optical Imaging Using CUBIC

Background

Tissue optical clearing (TOC) enables the visualization of regions of interest in deep tissue and is now widely established. In recent years, a variety of methods have been developed based on organic solvents, water-based non-gels, and hydrogels (Ertürk et al., 2011; Chung and Deisseroth, 2013; Ke et al., 2013); however, both the CLARITY and 3DISCO protocols are associated with significant drawbacks: 3DISCO loses endogenous protein fluorescence and involves toxic and flammable substances (Matsumoto et al., 2019), while CLARITY relies on dedicated equipment for electrophoresis. In addition, acrylamide has carcinogenicity and toxicity in its aqueous form (Hogervorst et al., 2007). Compared with these other brain-clearing techniques, CUBIC is safe, simple to operate, and efficiently achieves transparency. This method clears fixed brain samples in a few days to render the brain transparent, thus meeting the quality requirements of optical imaging of the sample. Using the standard CUBIC tissue clearing procedure, we subject mouse brain samples to transcardial perfusion, clearing, immersion in antibody solution, and refractive index matching. The aminoalcohol used can effectively remove hemochrome from hemoglobin and myoglobin, which not only reduces light scattering but also lessens the absorption of light to achieve further decolorization and transparency. When combined with rotating-disc confocal microscopy, CUBIC facilitates comprehensive and quantitative analyses for understanding the brain structure on the millimeter-scale level. It boasts a simple clearance procedure (Muntifering et al., 2018), excellent clearance effect in a short time frame (approximately 11 days) (Wan et al., 2018), and outstanding fluorescence preservation (Tainaka et al., 2018), making it a popular option among research labs.

After performing the CUBIC protocol, the tissues are optically transparent (Richardson and Lichtman, 2015), which meets the quality requirements for the optical imaging of samples. In addition, it is compatible with traditional immunofluorescence methods (Xu et al., 2019) and can be used by researchers to explore the effects of immunolabeling. Combined with confocal, two-photon microscopy or light-sheet microscopy, CUBIC can easily obtain high-resolution (millimeter level) 3D brain images (Isogai et al., 2017; Irie et al., 2018) to provide information about the type, position, and activities of cells and cellular networks (Tomer et al., 2014), promoting research on the relationship between brain structure and function. CUBIC provides an integrative platform to advance scalable and collaborative 3D image analysis (Matsumoto et al., 2019) that enables a wide range of users to perform experiments targeting cellular and organ layers with multiple samples (Mano et al., 2020). This technology can solve developmental and fundamental neuroscience problems (Mano et al., 2018) and answer scientific questions associated with Alzheimer's disease (AD) (Liebmann et al., 2016) and other neurodegenerative diseases (Ueda et al., 2020).