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


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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Figure 1: Overview of the CUBIC pipeline.
Figure 2: Procedure of the simple immersion protocol.
Figure 3: Procedure of the CB-perfusion protocol.
Figure 4: Whole-organ imaging with LSFM.
Figure 5: Preprocessing of acquired 3D image for comparison analysis.
Figure 6: Calculation of signal subtraction.


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We thank the lab members at RIKEN QBiC and The University of Tokyo, in particular S.I. Kubota for his kind help in preparing the materials; A. Millius for his critical reading and editing of the manuscript; and T. Mano for his kind contributions and suggestions to discuss image resolution. This work was supported by the Program for Innovative Cell Biology by Innovative Technology and the Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan; a Grant-in-Aid for Scientific Research (S) (grant No. 25221004), for Scientific Research on Innovative Areas (grant no. 23115006) and for Young Scientists (A) (grant no. 15H05650) from MEXT/Japan Society for the Promotion of Science (JSPS); by the strategic programs for R&D (President's discretionary fund) of RIKEN; by an intramural Grant-in-Aid from the RIKEN QBiC; by a grant from AMED-CREST; by the RIKEN Special Postdoctoral Research Program; by the RIKEN Foreign Postdoctoral Researcher Program; by a Grant-in-Aid from the Japan Foundation for Applied Enzymology; by the Brain Sciences Project of the Center for Novel Science Initiatives of the National Institutes of Natural Sciences (grant nos. BS261004 and BS271005); by the Tokyo Society of Medical Science; and by the Shimabara Science Promotion Foundation.

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H.R.U., E.A.S., K.T. and D.P. designed the study. E.A.S., H.Y. and A.K. performed most of the immersion protocol. K.T. performed most of the CB-perfusion protocol. D.P. performed most of the computational image analysis. A.K. developed the improved immersion protocol. All authors discussed the results and commented on the manuscript text.

Corresponding author

Correspondence to Hiroki R Ueda.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Data

Scripts and guide for software described. (ZIP 49 kb)


Almost all cells are roughly detected as single spots in such a relatively dense region. The analysis was performed with Imaris software. Some of parameters are manually adjusted. (MP4 26325 kb)

Spot-counting analysis of hippocampal neurons in Thy1-YFP-H Tg mouse brain shown in Fig. 4b-f.

Almost all cells are roughly detected as single spots in such a relatively dense region. The analysis was performed with Imaris software. Some of parameters are manually adjusted. (MP4 26325 kb)

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Susaki, E., Tainaka, K., Perrin, D. et al. Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. Nat Protoc 10, 1709–1727 (2015).

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