Advanced CUBIC tissue clearing for whole-organ cell profiling

Tissue-clearing techniques are powerful tools for biological research and pathological diagnosis. Here, we describe advanced clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) procedures that can be applied to biomedical research. This protocol enables preparation of high...

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Published inNature protocols Vol. 14; no. 12; pp. 3506 - 3537
Main Authors Matsumoto, Katsuhiko, Mitani, Tomoki T, Horiguchi, Shuhei A, Kaneshiro, Junichi, Murakami, Tatsuya C, Mano, Tomoyuki, Fujishima, Hiroshi, Konno, Ayumu, Watanabe, Tomonobu M, Hirai, Hirokazu, Ueda, Hiroki R
Format Journal Article
LanguageEnglish
Published England Nature Publishing Group 01.12.2019
Subjects
Animals
Biological research
Biomedical research
Brain
Brain - diagnostic imaging
Brain mapping
Brain research
Clearing
Coloring Agents
Computational neuroscience
Fluorescence
Fluorescent Dyes
Fluorescent proteins
Imaging, Three-Dimensional - methods
Indicators and Reagents
Medical imaging
Medical research
Medicine, Experimental
Methods
Mice
Neuroimaging
Neuroimaging - methods
Optical Imaging - methods
Organs
Reagents
Submerging
Tissue engineering
Viruses
Japan
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Summary:Tissue-clearing techniques are powerful tools for biological research and pathological diagnosis. Here, we describe advanced clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) procedures that can be applied to biomedical research. This protocol enables preparation of high-transparency organs that retain fluorescent protein signals within 7-21 d by immersion in CUBIC reagents. A transparent mouse organ can then be imaged by a high-speed imaging system (>0.5 TB/h/color). In addition, to improve the understanding and simplify handling of the data, the positions of all detected cells in an organ (3-12 GB) can be extracted from a large image dataset (2.5-14 TB) within 3-12 h. As an example of how the protocol can be used, we counted the number of cells in an adult whole mouse brain and other distinct anatomical regions and determined the number of cells transduced with mCherry following whole-brain infection with adeno-associated virus (AAV)-PHP.eB. The improved throughput offered by this protocol allows analysis of numerous samples (e.g., >100 mouse brains per study), providing a platform for next-generation biomedical research.
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ISSN:1754-2189
1750-2799
DOI:10.1038/s41596-019-0240-9