Chemical Landscape for Tissue Clearing Based on Hydrophilic Reagents.

Cell reports (2018-08-23)
Kazuki Tainaka, Tatsuya C Murakami, Etsuo A Susaki, Chika Shimizu, Rie Saito, Kei Takahashi, Akiko Hayashi-Takagi, Hiroshi Sekiya, Yasunobu Arima, Satoshi Nojima, Masako Ikemura, Tetsuo Ushiku, Yoshihiro Shimizu, Masaaki Murakami, Kenji F Tanaka, Masamitsu Iino, Haruo Kasai, Toshikuni Sasaoka, Kazuto Kobayashi, Kohei Miyazono, Eiichi Morii, Tadashi Isa, Masashi Fukayama, Akiyoshi Kakita, Hiroki R Ueda
ABSTRACT

We describe a strategy for developing hydrophilic chemical cocktails for tissue delipidation, decoloring, refractive index (RI) matching, and decalcification, based on comprehensive chemical profiling. More than 1,600 chemicals were screened by a high-throughput evaluation system for each chemical process. The chemical profiling revealed important chemical factors: salt-free amine with high octanol/water partition-coefficient (logP) for delipidation, N-alkylimidazole for decoloring, aromatic amide for RI matching, and protonation of phosphate ion for decalcification. The strategic integration of optimal chemical cocktails provided a series of CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis) protocols, which efficiently clear mouse organs, mouse body including bone, and even large primate and human tissues. The updated CUBIC protocols are scalable and reproducible, and they enable three-dimensional imaging of the mammalian body and large primate and human tissues. This strategy represents a future paradigm for the rational design of hydrophilic clearing cocktails that can be used for large tissues.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sodium citrate tribasic dihydrate, ACS reagent, ≥99.0%
Sigma-Aldrich
Mineral oil, light oil
Sigma-Aldrich
Anti-NeuN Antibody, clone A60, clone A60, Chemicon®, from mouse
Sigma-Aldrich
Histodenz, nonionic density gradient medium
Sigma-Aldrich
1,3-Cyclohexanebis(methylamine), mixture of isomers, 98%
Sigma-Aldrich
Monoclonal Anti-Glial Fibrillary Acidic Protein (GFAP)−Cy3 antibody produced in mouse, clone G-A-5, purified from hybridoma cell culture