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Representative Results






Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Published: June 30th, 2016



1Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, 2W.M. Keck Science Department, Claremont McKenna, Pitzer & Scripps Colleges

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2′-thiodiethanol (TDE), a previously described refractive index matching solution.

The ability to image complete neuroanatomical structures is immensely valuable for understanding the brain in health and disease. Traditionally, 3D imaging has required tissue sectioning to provide the axial resolution and to visualize deep anatomical structures. This approach can produce high-resolution data sets, but requires sophisticated image reconstruction techniques and is very labor intensive. As a result, it has been limited to the imaging of small volumes of tissue1-3. Optical sectioning, on the other hand, is well suited for the creation of high-resolution 3D images of fluorescently labeled tissues. Since optical sectioning is inherently three di....

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All experiments were performed in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines. THY1-YFP, PLP-EGFP and PV-TdTomato mice were used for these experiments, but any mice expressing fluorescent proteins can be successfully cleared and imaged.

1. Tissue Preparation

Caution: Paraformaldehyde (PFA) and acrylamide are toxic and irritants. Perform experiments utilizing these reagents in a fume hood and with appropriate personal protective equip.......

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Visualizing the structural organization of the brain is critical for understanding how morphology and connectivity affect brain function in health and disease. Optical clearing techniques make it possible to image cell populations in 3D in intact tissues, allowing us to study morphology and connectivity cohesively.

Mice that express fluorescent proteins driven by promoters specific for sub-populations of cells permit one to teas.......

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Passive clearing of hydrogel embedded tissues (passive CLARITY) is a simple and inexpensive method for clearing large pieces of tissue. This approach does not require dedicated equipment and can easily be performed in a temperature-controlled shaker. Over the span of a few weeks, even large, highly myelinated tissues, such as an entire brain or spinal cord, will become transparent and suitable for microscopy. Even though this report has focused on the clearing of CNS tissues, passive CLARITY can be applied to any tissue........

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This work was generously supported by the National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS) grant 1R01NS086981 and the Conrad N. Hilton Foundation. We thank Dr. Laurent Bentolila and Dr. Matthew Schibler for their invaluable assistance with confocal microscopy. We thank those that have contributed to the CLARITY forum ( We especially thank Dr. Karl Deisseroth for opening up his lab to teach this fascinating technique. The authors are grateful for the generous support from the Brain Mapping Medical Research Organization, Brain Mapping Support Foundation, Pierson-Lovelace Foundation, The....

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Name Company Catalog Number Comments
10X phosphate buffered saline (PBS) Fisher BP399-1 buffers
32% paraformaldehyde (PFA) Electron Microscopy Sciences 15714-5 perfusion and hydrogel
2,2'-thiodiethanol (TDE) Sigma-Aldrich 166782-500G refractive index matching solution
40% acrylamide Bio-Rad 161-0140 hydrogel
2% bis-acrylamide Bio-Rad 161-0142 hydrogel
VA-044 initiator Wako Pure Chemical Industries, Ltd. VA044 hydrogel
boric acid Sigma-Aldrich B7901 clearing buffer
sodium dodecyl sulfate (SDS) Fisher BP166-5 clearing buffer
sodium hydroxide Fisher 55255-1 buffers
sodium azide Sigma-Aldrich 52002-100G preservative
triton x-100 Sigma-Aldrich X100-500G buffers
heparin Sigma-Aldrich H3393-50KU perfusion
sodium nitrate Fisher BP360-500 perfusion
DAPI Molecular Probes D1306 nuclear stain
99.9% isoflurane Phoenix 57319-559-06 anesthetic
hydrocholoric acid Fisher A1445-500 buffers
glass bottom dish Willco HBSB-5040 Willco dishes
reusable adhesive Bostick 371351 Blu-Tack
silicon elastomer World Precision Instruments KWIK-CAST Kwik-Cast
lint-free wipe Kimberly-Clark 34120 KimWipe
mouse brain matrix Roboz SA-2175 sectioning tissue
matrix blades Roboz RS-9887 sectioning tissue
peristaltic pump Cole-Parmer 77122-24 pefusion
laser scanning confocal microscope Leica SP5 microscopy
imaging software Leica LAS AF microscopy

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