CUBIC protocol for whole-brain and whole-body imaging

CUBIC offers a fluorescent-preserving, high-performance and device-free tissue clearing method based on hydrophilic reagents. It enables reproducible whole-organ and whole-body clearing and rapid 3D imaging with LSFM. CUBIC also provides processing and analysis of 3D images for extracting biological information.
Therefore, CUBIC presents a platform of whole-organ/body imaging with single-cell resolution and image informatics, which enables a wide range of users to perform experiments targeting cellular and organ layers with multiple samples.

For further details, please refer to our publications:

1. E. A. Susaki, K. Tainaka, D. Perrin, F. Kishino, T. Tawara, T. M. Watanabe, C. Yokoyama, H. Onoe, M. Eguchi, S. Yamaguchi, T. Abe, H. Kiyonari, Y. Shimizu, A. Miyawaki, H. Yokota and H. R. Ueda (2014). Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell 157, 726–739.
2. K. Tainaka, S. I. Kubota, T. Q. Suyama, E. A. Susaki, D. Perrin, M. Ukai-Tadenuma, H. Ukai and H. R. Ueda (2014). Whole-body imaging with single-cell resolution by tissue decolorization. Cell 159, 911–924.
3. E. A. Susaki, K. Tainaka, D. Perrin, H. Yukinaga, A. Kuno and H. R. Ueda (2015). Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging with single-cell resolution. Nature Protocols 10, 1709–1727.

When using the CUBIC protocol, please make sure to cite the relevant publications: [1,3] for whole-brain imaging, [2,3] for whole-body imaging.


CUBIC clearing protocol with Reagent-1A
The CUBIC clearing protocol with Reagent-1A was developed for further fluorescent preservation, and was mentioned in our recent review: E. A. Susaki and H. R. Ueda (2016), Whole-body and whole-organ clearing and imaging techniques with single-cell resolution: Toward organism-level systems biology in mammals, Cell Chemical Biology.
For details on this protocol, please refer to this file (deposited January 6th, 2016).


Analysis software
The latest version of the tools for image analysis described in [3] are available on GitHub.
Click here to access the repository.


Sample files
To reproduce the analysis in described in [3], you can download the raw images for the Light +/- Arc-dVenus dataset and the atlas file used for registration.
Click on the links below to download the tgz archives. Please note that these files are very large.

• Brain 1, DV imaging, nuclear counterstaining (4.9 GB).
• Brain 1, VD imaging, nuclear counterstaining (5.2 GB).
• Brain 1, DV imaging, Arc-dVenus channel (4.6 GB).
• Brain 1, VD imaging, Arc-dVenus channel (4.9 GB).
• Brain 2, DV imaging, nuclear counterstaining (5.0 GB).
• Brain 2, VD imaging, nuclear counterstaining (5.1 GB).
• Brain 2, DV imaging, Arc-dVenus channel (4.7 GB).
• Brain 2, VD imaging, Arc-dVenus channel (4.8 GB).
• Brain 3, DV imaging, nuclear counterstaining (5.1 GB).
• Brain 3, VD imaging, nuclear counterstaining (5.3 GB).
• Brain 3, DV imaging, Arc-dVenus channel (4.9 GB).
• Brain 3, VD imaging, Arc-dVenus channel (5.1 GB).
• Brain atlas (44 MB).

Additional sample files

• Susaki-NatCommun(2020)_Thy1-YFP-H_Brain_VD_YFP (5.4 GB).
• Susaki-NatCommun(2020)_B6WT_Brain_DV_NeuN_Immunostaining-channel (1.2 GB).
• Susaki-NatCommun(2020)_B6WT_Brain_DV_NeuN_Nuclear-staining-channel (1.3 GB).
• Susaki-NatCommun(2020)_B6WT_Brain_DV_PV_Immunostaining-channel (0.8 GB).
• Susaki-NatCommun(2020)_B6WT_Brain_DV_PV_Nuclear-staining-channel (1.2 GB).

Updated CUBIC-HistoVIsion (CUBIC-HV1.1) and representative data
We somewhat modified the original CUBIC-HistoVIsion (HV) 1.0 protocol reported in Susaki et al. (2020) to improve the signal-and-noise ratio. A set of z-stack images of the whole ICR mouse brain stained by CUBIC-HV1.1 with anti-NeuN, A555-conjugated antibody (Merck Millipore #MAB377A5) is provided below.

Susaki-NatCommun(2020)_Supplementary_HV1.1_IcrWT_Brain_DV_NeuN-A555_Immunostaining-channel (1.6 GB).

Document

Gel embedding of CUBIC-R-cleared sample
This movie explains how to embed the CUBIC-R-cleared sample in CUBIC-R/agarose, documented in Matsumoto et al. Nature Protocol (2019) and Susaki et al. Nature Communications (2020).

• Gel embedding of CUBIC-R-cleared sample