PROTOCOL Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging Etsuo A Susaki1–3,7, Kazuki Tainaka1–3,7, Dimitri Perrin4,7, Hiroko Yukinaga3, Akihiro Kuno1,2,5,6 & Hiroki R Ueda1–3 1Department of Systems Pharmacology, The University of Tokyo, Tokyo, Japan. 2Japan Agency for Medical Research and Development (AMED) – Core Research for Evolutionary Science and Technology (CREST), AMED, Tokyo, Japan. 3Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center (QBiC), Osaka, Japan. 4School of Electrical Engineering and Computer Science, Science and Engineering Faculty, Queensland University of Technology (QUT), Brisbane, Queensland, Australia. 5Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan. 6PhD Program in Human Biology, School of Integrative and Global Majors, University of Tsukuba, Ibaraki, Japan. 7These authors contributed equally to this work. Correspondence should be addressed to H.R.U. (
[email protected]). Published online 8 October 2015; doi:10.1038/nprot.2015.085 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.