Format

Send to

Choose Destination
Nat Protoc. 2019 Dec;14(12):3506-3537. doi: 10.1038/s41596-019-0240-9. Epub 2019 Nov 20.

Advanced CUBIC tissue clearing for whole-organ cell profiling.

Author information

1
Laboratory for Synthetic Biology, RIKEN Center for Biosystems Dynamics Research, Osaka, Japan.
2
Faculty of Medicine, Osaka University, Osaka, Japan.
3
Osaka University Hospital, Osaka, Japan.
4
Department of Electrical Engineering and Information Systems, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan.
5
Laboratory for Comprehensive Bioimaging, RIKEN Center for Biosystems Dynamics Research, Osaka, Japan.
6
Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
7
Department of Information Physics and Computing, Graduate School of Information Science and Technology, The University of Tokyo, Tokyo, Japan.
8
International Research Center for Neurointelligence (WPI-IRCN), UTIAS, The University of Tokyo, Tokyo, Japan.
9
Department of Neurophysiology & Neural Repair, Gunma University Graduate School of Medicine, Maebashi, Japan.
10
Laboratory for Synthetic Biology, RIKEN Center for Biosystems Dynamics Research, Osaka, Japan. uedah-tky@umin.ac.jp.
11
Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. uedah-tky@umin.ac.jp.
12
Department of Information Physics and Computing, Graduate School of Information Science and Technology, The University of Tokyo, Tokyo, Japan. uedah-tky@umin.ac.jp.
13
International Research Center for Neurointelligence (WPI-IRCN), UTIAS, The University of Tokyo, Tokyo, Japan. uedah-tky@umin.ac.jp.

Abstract

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.

PMID:
31748753
DOI:
10.1038/s41596-019-0240-9

Supplemental Content

Full text links

Icon for Nature Publishing Group
Loading ...
Support Center