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Nat Protoc. 2015 Nov;10(11):1709-27. doi: 10.1038/nprot.2015.085. Epub 2015 Oct 8.

Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging.

Susaki EA1,2,3, Tainaka K1,2,3, Perrin D4, Yukinaga H3, Kuno A1,2,5,6, Ueda HR1,2,3.

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Department of Systems Pharmacology, The University of Tokyo, Tokyo, Japan.
Japan Agency for Medical Research and Development (AMED) - Core Research for Evolutionary Science and Technology (CREST), AMED, Tokyo, Japan.
Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center (QBiC), Osaka, Japan.
School of Electrical Engineering and Computer Science, Science and Engineering Faculty, Queensland University of Technology (QUT), Brisbane, Queensland, Australia.
Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan.
PhD Program in Human Biology, School of Integrative and Global Majors, University of Tsukuba, Ibaraki, Japan.


Here we describe a protocol for advanced CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis). The CUBIC protocol enables simple and efficient organ clearing, rapid imaging by light-sheet microscopy and quantitative imaging analysis of multiple samples. The organ or body is cleared by immersion for 1-14 d, with the exact time required dependent on the sample type and the experimental purposes. A single imaging set can be completed in 30-60 min. Image processing and analysis can take <1 d, but it is dependent on the number of samples in the data set. The CUBIC clearing protocol can process multiple samples simultaneously. We previously used CUBIC to image whole-brain neural activities at single-cell resolution using Arc-dVenus transgenic (Tg) mice. CUBIC informatics calculated the Venus signal subtraction, comparing different brains at a whole-organ scale. These protocols provide a platform for organism-level systems biology by comprehensively detecting cells in a whole organ or body.

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