Abstract
The development, refinement, and use of techniques that allow high-throughput imaging of whole brains with cellular resolution will help us understand the complex functions of the brain. Such techniques are crucial for the analysis of complete neuronal morphology-anatomical and functional-connectivity, and repeated molecular phenotyping. CLARITY is a recently introduced technique that produces structurally intact, yet optically transparent tissue, which may be labeled and imaged without sectioning. However, the utility of this technique depends on several procedural variables during the process in which the light-scattering lipids in a tissue are replaced by a transparent hydrogel matrix. Here, we systematically varied a number of factors (including temperature, hydrogel composition, and polymerization conditions) to provide an optimized, highly replicable CLARITY procedure for clearing mouse brains. We found that for these preparations optimal tissue clearing requires electrophoresis (and cannot be achieved with passive clearing alone) for 5 d with a combination of 37 and 55°C temperature. Although this protocol is optimized for brains, we also show that it can be used to clear and analyze a variety of organs. Brain or other tissue prepared using this protocol is suitable for high-throughput imaging with confocal or single-plane illumination microscopy.
Highlights
Understanding brain function is a fundamental scientific objective with crucial clinical relevance
Electrophoresis decreases the amount of time required to produce clear tissue
At 37°C, moderately clear tissue can be achieved in 1 month
Summary
Understanding brain function is a fundamental scientific objective with crucial clinical relevance. Realization of this goal is hampered by the complexity of the brain. There are roughly 86 billion neurons in the human brain (75 million in the mouse) and these neurons interact in complex neural circuits. Using Camillo Golgi’s newly developed techniques to label neurons and their processes, Santiago. Received March 4, 2015; accepted April 29, 2015; First published May 8, 2015. 2J.R.E. and Y.N. performed the clearing and imaging experiments. J.R.E., K.D., S.A.J., and P.W.F. conceived the experiments and wrote this manuscript
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