[Abstract] Here, we describe a simple and efficient tissue clearing method, CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis), which was first introduced in 2014 (Susaki et al ., 2014). In the present study, the hemispheres of wild-type adult mice were cleared by standard procedures and were browning free with good transparency and no significant changes in tissue shape or volume over a short time frame. Further, in combination with immunofluorescence staining, 3D high-resolution images of tyrosine hydroxylase-positive dopaminergic neurons in the substantina striatum region were obtained by rotating-disc confocal microscopy, in addition to images of mouse cortical and hippocampal neurons. Considering the limitations of existing brain tissue removal methods, such as CLARITY, 3DISCO, and SeeDB, CUBIC boasts simple operation, high fluorescent protein preservation, and good antibody penetration. Moreover, CUBIC variants and optimizations are available for the 3D imaging of rodent, large primate, and human tissues to meet researchers' specific requirements, which could make it a popular option among research labs. Our study demonstrates the use of the CUBIC protocol for understanding brain structure and function in model animals with neurological diseases. Flowchart of the CUBIC clearing and imaging protocol. A. Tissue preparation. The adult mouse was first perfused with cold PBS/PFA solution. The harvested brain tissue was fixed with 4% PFA on day 0. B. Tissue clearing. The tissue was incubated with 1/2-water-diluted CUBIC-1, resulting in the removal of hemochrome and lipid membranes. It usually takes 1-6 days to become more transparent. C. Immunostaining. Tissues (2-mm brain slice) were blocked and then stained with directly conjugated anti-TH diluent (1:100) and FITC diluent (1:500) on days 7-9. D. RI matching. Tissues were immersed in 1/2-PBS-diluted CUBIC-2 on day 10 to match the refractive index (RI). Following immersion in imaging oil for 2 h, the cellular position within tissues was photographed by confocal microscopy.

连响 罗

Xiaoyu Chen

Ruijian Wu

Han Wang

Hui Luo

BIO-PROTOCOL

Bio-protocol

Clearing of the Mouse Brain for Optical Imaging Using CUBIC

The hemispheres of wild-type adult mice were cleared by standard procedures and were browning free with good transparency and no significant changes in tissue shape or volume over a short time frame in the CUBIC method.

Clearing of the Mouse Brain for Optical Imaging Using CUBIC

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Journal Info

Category	Quartile
BIOLOGY	3