Methods/New Tools

Novel Tools and Methods

Optimization of CLARITY for Clearing Whole-Brain
and Other Intact Organs1,2,3
Jonathan R. Epp,1,2,3 Yosuke Niibori,1,2,3 Hwa-Lin (Liz) Hsiang,1,2,3 Valentina Mercaldo,1,2,3
Karl Deisseroth,4 Sheena A. Josselyn,1,2,3 and Paul W. Frankland1,2,3
DOI:http://dx.doi.org/10.1523/ENEURO.0022-15.2015
1

Program in Neurosciences and Mental Health, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada,Institute of Medical Sciences, University of Toronto, Toronto, Ontario M5G 1X8, Canada, 3Departments of
Psychology and Physiology, University of Toronto, Toronto, Ontario M5G 1X8, Canada, and 4Department of
Bioengineering and Psychiatry, Stanford University, Stanford, California 94305
2

Abstract
The development, refinement, and use of techniques that allow high-throughput imaging of whole brains withcellular 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 techniquedepends 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 andanalyze 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.
Key words: 3D imaging; CLARITY; light sheet microscopy; neuron morphology; tissue clearing; whole-brain
imaging

Significance Statement
CLARITY is a novel technique for optical clearance and intact imaging of biological specimens. This method
was introduced in 2013 but has been difficult for some researchers to implement. Here, we have optimized
the technique to increase the success and consistency of the clearing procedure. Using our protocol, it ispossible to clear not only brain, but other tissues as well. In addition, the cleared tissue becomes sufficiently
transparent such that no specialized microscopes are necessary to visualize the sample. This optimizationof CLARITY will make the technique more widely accessible.

Introduction
Understanding brain function is a fundamental scientificobjective with crucial clinical relevance. Realization of thisReceived March 4, 2015; accepted April 29, 2015; First published May 8, 2015.
1
The authors declare no competing financial interests.
2
J.R.E. and Y.N. performed the clearing and imaging experiments. H.-L.H.

May-June 2015, 2(3) e0022-15.2015 1–15

goal is hampered by the complexity of the brain. There areroughly 86 billion neurons in the human brain (75 million in
the mouse) and these neurons interact in complex neuralcircuits. Using Camillo Golgi’s newly developed techniques to label neurons and their processes, Santiago
preformed stereotaxic surgeries. V.M. performed the BCA analysis. J.R.E.,
K.D., S.A.J., and P.W.F. conceived the experiments and wrote this manuscript.

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Table 1 List of equipment and reagents
Incubating orbital shaker
Vibrating tissue slicer
Recirculating water bathElectrophoresis power supply
Light sheet microscope
Laser-scanning confocal microscope
Wide-field epifluorescent microscope
Vacuum canister
SDS
Boric acid
Paraformaldehyde
Acrylamide solution (40%)
Bis-acrylamide solution (2%)
VA-044Glycerol
Triton-X 100
Sodium azide
10 N NaOH
Sodium phosphate monobasic anhydrous
FocusClear
Nitrogen gas
Propidium iodide
Clear tubing (PVC, chemical resistant)
Clear tubing (PVC, chemical resistant)Barbed tube fittings (polypropylene)
Barbed tube fittings (polypropylene)
Barbed tube fittings (polypropylene)
Barbed tube fittings (polypropylene)
Barbed tube fittings (polypropylene)
Manifold (polypropylene)
Manifold plugs (polypropylene)
Quick disconnect barbed plug
Quick disconnect barbed socket
Filter case
Filter cartridge
3M Scotch-weld Epoxy DP270
Platinum wire
Nalgene 60 ml wide-mouth jar
Cell strainer
Paraffin embedding cassetteRamon y Cajal was among the first to use microscopy tounderstand brain function. Imaging studies in the traditionof Cajal have been plagued by either poor spatial scale(examining only a small part of a larger brain network) orpoor spatial resolution (examining a large network but atthe cost of understanding microstructure). The brain has ahigh lipid content, which makes it opaque and limits thedepth of light penetration. Light that does penetrate brain
3
This work was supported by Canadian Institute of Health Research grants
to P.W.F. (MOP-86762) and S.A.J. (MOP-74650). J.R.E. received funding froma National Alliance for Research on Schizophrenia and Depression Young
Investigator Grant.
Correspondence should be addressed to either Paul Frankland or Sheena A.
Josselyn, Program in Neurosciences and Mental Health, Hospital for Sick
Children, Ontario M5G 1X8, Canada, E-mail: paul.frankland@sickkids.ca or
sheena.josselyn@sickkids.ca.
DOI:http://dx.doi.org/10.1523/ENEURO.0022-15.2015
Copyright © 2015 Epp et al.
This is an open-access article distributed under the terms of the Creative
Commons Attribution 4.0 International, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is
properly attributed.

May-June 2015, 2(3) e0022-15.2015

VWR
Leica
VWR
Bio-Rad
LaVision Biotec
Zeiss
Nikon
McMaster Carr
Sigma
Sigma
Sigma
Bio-Rad
Bio-Rad
WAKO
Sigma
Sigma
Sigma
Sigma
Sigma
CelExplorer Labs
Praxair
Life Technologies
McMaster Carr
McMaster Carr
McMaster Carr
McMaster Carr
McMaster Carr
McMaster Carr
McMaster Carr
McMaster Carr
McMaster Carr
McMaster Carr
McMaster Carr
McMaster Carr
McMaster Carr
McMaster Carr
Alfa Aesar
VWR
Fisher
VWR

97027-346
VT 1200S
89203-010
PowerPac HC
UltraMicroscope
LSM 710
Eclipse 80i
2204K7
L3771
B6768
441244161-0140
161-0142
877-714-1920
G5516
T8787
S2002
656054
S8282
FC-102
P3566
5103K365103K34
53415K207
5463K245
5463K2458
5463K2457
5117K51
5364K231
4515K209
5154K6951545K63
4448K35
4422K61
7467A1710286
36319-547
22363548
18000-000

tissue is scattered, producing out-of-focus images inwhich the point source of the light cannot be localized.
Therefore, the high lipid content of the brain makes it
especially difficult to image at the mesoscale.
To overcome this obstacle, most experiments involveslicing the brain into relatively thin sections (20-50 ␮m)
before histological staining, imaging, and quantification.
However, sectioning the brain has its own drawbacks. For
instance, light continues to be scattered even within relatively thin brain sections (limiting axial resolution), sectioning may damage and/or distort the tissue, and muchof the rich structural information may be lost in sectioned
tissue, even if attempts are made to accurately reconstruct 3D images. For example, sectioned brain tissue
may not capture the full extent of neuronal projections(which may extend hundreds of micrometers), thus providing an incomplete view of neuronal branching andregional variations in spine density. One way to address
this limitation involves block-face imaging in which a microtome is coupled to a microscope. A tiled image of theeNeuro.sfn.org

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brain is acquired prior to cutting each section to produce
a 3D reconstruction of the intact tissue (Oh et al., 2014;
Vousden et al., 2014). This technique is accompanied bydifferent limitations in that it does not permit immunolabeling of the tissue and requires substantial effort to alignthe sections after imaging. An ideal method to interrogate
tissue is to microscopically examine intact, rather thansectioned, tissue.
Several tissue-clearing protocols have been describedto overcome the challenges of imaging thick tissue sections [including SCALE (Hama et al., 2011) and CUBIC(Susaki et al., 2014)] or combinations of organic solvents
such as benzyl alcohol/benzyl benzoate (Dodt et al., 2007)
or dibenzyl ether (Erturk et al., 2012). Although thesemethods yield tissue with varying degrees of transparency, they are accompanied by their own limitations. For
example, the clearing agents used in these protocols may
quench fluorescent proteins and these techniques do not
permit antibody labeling. However, a recent techniquecalled lipid-exchanged, anatomically rigid, imaging/immunostaining compatible, tissue hydrogel (CLARITY; Chunget al., 2013) overcomes these limitations to produce transparent tissue without quenching endogenous fluorescence, and this tissue can also be labeled via
immunohistochemistry.
CLARITY uses a strong detergent, SDS, to remove
lipids from the tissue. To preserve protein integrity and the
3D structure of the tissue, tissue is first perfused with aformaldehyde and acrylamide solution that forms structural hydrogel scaffolding. With the aid of an electrophoretic current, SDS then removes the lipids that are notlinked to the scaffold. Cleared tissue may then be labeled
and/or imaged with substantially increased light penetration and high-resolution images may be collected several
millimeters or more below the tissue surface.
The CLARITY protocol was originally published by the
Deisseroth lab (Chung et al., 2013) and methods to optimize
the imaging component subsequently added (Tomer et al.,2014). A modified CLARITY protocol, using only passive

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clearing techniques to avoid the possible tissue damage
produced by electrophoretic current, was also recently described (Tomer et al., 2014; Yang et al., 2014; Zhang et al.,2014). However, to effectively take advantage of CLARITY, it
is crucial to obtain brains that are highly transparent (fordeep imaging) and rigid (to facilitate registration to a reference atlas). While passive clearing may produce transparent
tissue, electrophoresis is necessary to achieve the full potential of CLARITY. Here we describe a revised CLARITY
protocol designed to fully optimize the clearing process
while minimizing concerns related to tissue damage anddistortion. Tissue cleared with this protocol can be imaged
with virtually any fluorescent microscope, making the technique widely accessible. In addition, we show that our optimized protocol may be used to image numerous intact
organs in addition to the brain, with minimal modification.

Materials and Methods
All of the necessary components and suppliers for performing the procedure can be found in Table 1.
Mice
Adult (6-8 week) WT female mice in a hybrid (F1) genetic
background were used for most studies (C57BL/6NTac ⫻129S6/SvEvTac). In some studies, we also used maleand female activity-regulated cytoskeleton-associatedprotein-targeted recombination (ArcTRAP) in active populations of mice (Guenthner et al., 2013; Jax, B6.129(Cg)Arctm1.1(cre/ERT2)Luo/J) crossed with a fluorescent reporter
strain. In the ArcTRAP mice, the tamoxifen-inducible CreERT2 protein is driven by endogenous promoter/enhancer elements of the activity-dependent immediate early gene Arc(Lyford et al., 1995). ArcTRAP mice were crossed with a reporter mouse (Jax, B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J) in which loxP sequences flank a STOP cassette upstreamof a tdTomato reporter. In this way, following systemic tamoxifen injection, cells that are sufficiently active will show
Cre-mediated recombination, thereby removing the STOP
cassette and allowing expression of tdTomato. A 10 mg/ml

Figure 1 Schematic of electrophoresis and imaging components. Schematic of the electrophoresis system used for active clearingof tissue. A, A recirculating water bath filled with clearing solution is connected via chemical- and heat-resistant tubing to a filter unit (whichremoves particulate matter and reduces bubbles in the clearing solution). The clearing solution is then split into six parallel tube lines, eachcontaining one ETC chamber. The fluid output of the chambers is then recombined into a single line before entering the recirculator. Powersupplies provide current to each ETC chamber. Schematic of the LaVision Biotec UltraMicroscope (B) compared with confocal microscope (C).
The light sheet design permits high-speed image capture of large tissue volumes. In contrast, a confocal microscope may be used forhigh-resolution imaging of CLARITY tissue, although the acquisition rate is much slower.

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solution of 4-hydroxytamoxifen was prepared by first dissolving the drug in 100% ethanol and then suspending thesolution in sunflower oil. Mice were injected with a dose of
20 mg/ml. Genotypes were determined by PCR analysis of
tail DNA samples as previously described (Guenthner et al.,2013). Mice were bred in-house and group housed (3-5 mice
per cage) on a 12 h light/dark cycle with food and water
available ad libitum. Behavioral experiments were conducted during the light phase of the cycle. All procedures
were conducted in accordance with the policies of the institutional Animal Care and Use Committee and conformed to
the federal guidelines on the care and use of laboratory
animals.
Hydrogel solution preparation
The hydrogel solution used to perfuse the tissue iscomposed of four key reagents: acrylamide, bisacrylamide, formaldehyde, and a thermal initiator (VA-044)
in PBS. Different strengths of hydrogels can be producedby varying the concentrations of these reagents. We began with the original hydrogel solution (referred to here as
4:4:0.05) containing final concentrations of 4% acrylamide, 0.05% bis-acrylamide, and 4% formaldehyde. We
first prepared a 16% formaldehyde solution by adding 80
g of paraformaldehyde powder to 400 ml of dH2O andheated the solution to 60-65°C. The solution was clearedwith several drops of 10 N NaOH and the volume wasfilled to 500 ml. Finally, we filtered the solution and cooled
to 4°C. Next, we combined 40 ml of cold 40% acrylamide,
10 ml of cold 2% bis-acrylamide, 40 ml of 16% formaldehyde, 40 ml of 10⫻ PBS, and 220 ml of cold dH2O and,
finally, 1 g of VA-044 thermal initiator. The solution was
kept on ice during preparation and was either used immediately after preparation or divided into 40 ml aliquots
and frozen at ⫺20°C for later use. Modified hydrogel
solutions were made following the same protocol but with
3% final concentrations of acrylamide and formaldehyde
or 0.025% bis-acrylamide.

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to wash residual hydrogel solution out of the tissue. The
tissue-clearing solution was prepared by dissolving 61.83
g of boric acid and 200 g of SDS in 4.5 L of dH2O. The final
pH was adjusted to 8.5 using 10 N NAOH.
Active tissue clearing
We used a modular multichamber design that allowed
six brains to be cleared simultaneously using a single
recirculator (Fig. 1A). Electrophoretic tissue-clearing (ETC)
components were connected in parallel to the recirculator
with temperature- and chemical-resistant tubing. Clearing
solution from the recirculator was first passed through afilter cartridge and then into a hub that divided the solution
into six tubing lines connected to the ETC chambers thatin turn were connected to another hub that connects backto the recirculator. Power supplies were connected to theplatinum leads of the ETC chambers with alligator clips.

Tissue collection for brain samples
Mice were transcardially perfused with ice-cold PBS
followed by 25 ml of cold hydrogel solution. The brains
were rapidly extracted and submersed in cold hydrogel
solution and postfixed in the dark at 4°C for 24 h in 10 mlof the same hydrogel solution.
Tissue polymerization
To ensure consistent hydrogel polymerization, we removed oxygen from the hydrogel solution by placing thesample vials in a vacuum chamber for 30 min. After
vacuuming, the chamber was flooded with pure nitrogengas to displace oxygen as the chamber was opened andthe lids placed on the sample vials. Polymerization of thehydrogel solution depends on the VA-044 thermal initiator, which is triggered by tissue warmed to 37°C. Therefore, we placed the sealed sample tubes in a rotating
incubator at 37°C for 3 h. Once the hydrogel was polymerized, excess gel was removed from the brain by gently
wiping the gel from the surface with a Kimwipe. We thenplaced the cleaned brain into a new 50 ml tube containing
clearing solution (4% SDS in 0.2 M borate buffer) overnight
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Figure 2 Active clearing of brains with ETC is more efficient thanpassive clearing. A, Passive clearing is temperature dependent withvery little clearing at 21°C occurring after 1 month in SDS. At 37 and
55°C the brain becomes progressively clearer after 1 month in SDS but
does not get as clear as with active (ETC) clearing. B, Relative to freshly
fixed and uncleared tissue, protein content is well preserved following
both active and passive clearing methods, regardless of temperatureused. Data are shown relative to brains that were perfused and polymerized with the same hydrogel solution but were not cleared. Olfactory bulb tissue from each clearing condition was homogenized anda BCA assay was performed to estimate protein concentration. There
were no significant differences in protein concentration as an effect ofactive versus passive clearing (p ⫽ 0.4) or clearing temperature (p ⫽0.24).

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Figure 3 Clearing conditions for optimal transparency. A, Comparison of ETC temperature and hydrogel composition on tissuetransparency and expansion. There were significant main effects of clearing temperature (F(2,24) ⫽ 178.9, p ⬍ 0.00001) and hydrogel
concentration (F(3,24) ⫽ 16.2, p ⬍ 0.00001) on transparency Tissue appears clearest with 37/55°C ETC and 3% acrylamide, 3%
formaldehyde, and 0.025% hydrogel. Tissue expansion is less in the 37/55°C condition compared with either 37°C or 55°C alone. B,
Both 55 and 37/55°C produce tissue that is more transparent than 37°C, regardless of hydrogel composition (ps ⫽ 0.0002 and 0.0009,
respectively). Transmission was measured on a light table under standardized illumination conditions. A reference measurement wasmade without a sample and was set to 100%. The sample was then imaged on the light table and the percentage light intensity
through the specimen was recorded as a percentage of the reference value. C, Brains cleared with the combined 37/55°C clearing
protocol expand more in lower concentration hydrogel (3% formaldehyde:3% acrylamide:0.025% bis-acrylamide compared withhigher concentration hydrogel (4% formaldehyde:4% acrylamide:0.05% bis-acrylamide). The conditions for producing an optimal
combination of stable and clear tissue is to polymerize the tissue with hydrogel composed of 4% acrylamide, 4% formaldehyde, and0.05% bis-acrylamide and then clear the tissue with 5 d of ETC (4 d at 37°C and 1 d at 55°C).

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Table 2: Data analysis
Data
BCA analysis

Test
Main effects ANOVAnThree per group

Transparency

Two-way ANOVA

Three per group

Newman–KeulsPost hoc

Three per group

Main effect
Active versus
passive temperature
HydrogelTemperature
Interaction
Hydrogel 3:3:0.025
versus 3:3:0.05 3:3:0.025
versus 4:4:0.025 3:3:0.025
versus 4:4:0.05 3:3:0.05
versus 4:4:0.05
Temperature
37°C versus 37/55°C
37°C versus 55°C
37:55°C versus 55°C

F
0.79
1.68
16.20
178.90
0.70

P
0.40
0.24
0.000006
0.000000
0.63
0.00470.000230.000170.00530.000150.000093
0.00013

Figure 4 The effect of ETC duration and temperature on imaging depth. A 2mm thick coronal section before (A) and after (B) clearing with out
optimized protocol. C, Coronal mouse brain section (2 mm thick) cleared with ETC for 1 d, 3 d, or 5 d at 37°C or for 4 d at 37°C plus an additional
day at 55°C. Sections were subsequently stained with propidium iodide and imaged using a confocal microscope under identical excitation
conditions without the use of Z-attenuation correction. There is an increase in depth at which a fluorescent signal may be imaged with respect
to clearing protocol. The 37/55°C clearing temperature provides deepest imaging penetration.

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Small pieces of tissue (such as 2 mm sections or dissected brain regions) were placed into a closed paraffin
embedding cassette. Larger tissues were placed into amesh cell strainer. The cell strainer or cassette was thenplaced between the electrodes and shielded from directcontact with the electrodes by two sheets of plastic mesh.
Various settings were used for tissue clearing. We haveexperienced difficulties in regulating the temperature and,as a result, maintaining tissue quality if the clearing voltage is too high or if the recirculator flow rate is too low.
Therefore, in all conditions we used a low voltage of 20 V
and a high flow rate of 30 L/min (5 L/min/ETC chamber).
Under these conditions, the temperature of the clearing
solution measured between the electrodes did not fluctuate from the set point. We then varied the temperature
between 21 and 55°C and length of clearing from 1 to 5 d
to produce the best combination of rigid and clear tissue.
The pH of the clearing solution was monitored daily andreplaced once the pH dropped from 8.5 to 8.0. After ETC
the tissue was rinsed in PBST for several days.
Passive tissue clearing
For experiments in which tissue was passively cleared weused the same clearing solution and kept the samples ona shaker at 21°C, 37°C, or 55°C. The clearing solution was

7 of 15exchanged every 3 d for up to 2 months. The tissues werethen rinsed in several changes of PBST for several days.
Tissue labeling
Propidium iodide labeling was performed by incubating
the tissue for 24-48 h at 37°C in a 1:2000 dilution of
propidium iodide in 0.1 M PBST. Immunohistochemistry
was performed using 1:100 dilutions of antibody in PBST
and incubating the tissues for 7 d at 37°C for the primary
antibody and an additional 7 d for the secondary antibody. The tissue was rinsed in 0.1 M PBST after eachantibody incubation. The antibodies used were mousemonoclonal anti-smooth muscle actin (Santa Cruz Biotechnology; catalog #SC-53142), rabbit polyclonal anticollagen type IV (Millipore; catalog #AB756P), rabbit
polyclonal anti-CD34 (Santa Cruz Biotechnology; catalog#SC-9095), goat anti-mouse Alexa 488 (Life Technologies; catalog #A11001), and goat anti-rabbit Alexa 488(Life Technologies; catalog #A11034).
Refractive index matching
After the PBST washes, the tissue was transferred toeither 80% glycerol or FocusClear. The tissue was left toequilibrate in the refractive index matching solution for
24 h at 37°C for glycerol or at room temperature for

Figure 5 Imaging depth optimization. Using ArcTRAP-tdTomato mice we visualized the endogenous fluorescence signal in micebrains that underwent 5 d of ETC at 37°C (A) or were cleared for 4 d at 37°C followed by 1 d at 55°C (B). Improved imaging depth
is observed using the combined 37/55°C protocol, suggesting that the improvement relates to light rather than dye penetration.

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FocusClear. Finally, the samples were then mounted in
the same medium for imaging.
Imaging setup
In principle, any type of fluorescent microscope can beused to image CLARITY-cleared tissue. However, themicroscope must be able to physically accommodate
the size of the sample and the stage translation, and theworking distance of the microscope objective should
be appropriate. A microscope should also allow images tobe acquired in a practical length of time. For these reasons, we routinely used light sheet microscopy (LaVision
BioTec UltraMicroscope; Fig. 1B) for acquisition of large
tissues. We also utilized a laser-scanning confocal microscope (Zeiss LSM710; Fig. 1C) equipped with 10⫻ NA 0.3
and 25⫻ NA 0.8 objectives and an Epifluorescent microscope (Nikon Eclipse 80i) with a 10⫻ NA 0.3 objective
Image analysis
We used a Lenovo ThinkStation D30 with 2⫻ Intel Xeon
3.1 GHz processors, a QuadK5000 4 GB graphics card,and 128 GB of RAM. For the light sheet microscope,
images were acquired using the ImSpector software suite(LaVision Biotec), confocal images were acquired using
Zen (Zeiss), and wide-field images were acquired with NIS
Elements (Nikon). Image processing was then performed
using a combination of software packages including
Imaris (Bitplane) for 3D reconstruction, ImageJ for 2D
stitching and basic adjustments, and AutoQuant (Media
Cybernetics) for deconvolution.

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Statistical analysis
Data shown in Figures 2 and 3 represent the mean ⫾SEM. Analyses were conducted using two-way ANOVAs
and Newman–Keuls post hoc tests as described inTable 2.

Results
Active versus passive tissue clearing
Tissue may be cleared passively or facilitated with electrophoresis (ETC) using custom electrophoretic chambers. Electrophoresis decreases the amount of timerequired to produce clear tissue. Passive clearing is lessexpensive as no additional equipment is required. In
terms of effectiveness of the clearing process, our comparisons demonstrate that although passive clearing canbe somewhat effective, it is not as effective as ETC.
Temperature remains a critical factor in passively clearing tissue. The efficacy of the clearing procedure increases with temperature. At 21°C, passive clearing has
little effect even after 1 or 2 months in SDS solution.
However, at 37°C, moderately clear tissue can beachieved in 1 month. At a higher temperature of 55°C,
very clear tissue can be produced in 1 month. However,after 1 month, passively cleared tissue is not nearly as
clear as tissue cleared with ETC (Fig. 2). Faster passive
clearing times may be achieved by using a lower concentration of acrylamide, formaldehyde, or bis-acrylamide,
but at the potential cost of decreased tissue preservationand increased tissue distortion.

Figure 6 Examining effects of different imaging media (FocusClear vs glycerol). Refractive matching of clarified tissue with either FocusClear or
glycerol produces equally transparent tissue, and high-quality images can be collected using either medium. C, D, We imaged the tdTomato signal
in the same sections shown in A and B. Excellent image clarity is produced whether the tissue is placed in FocusClear or 80% glycerol.

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Figure 7 continued
even a very basic epifluorescent microscope. In this case, aNikon Eclipse 80i with a mercury arc lamp was used to image a
2-mm-thick cleared section. A–F, Single image planes at 100 ␮mintervals through the z-stack. Bright and sharp cells can be seen
through at least 600 ␮m of the tissue.

Optimal hydrogel composition
The basic hydrogel solution is composed of formaldehyde (to act as a fixative), acrylamide (to form thehydrogel), bis-acrylamide (to act as a cross-linker), andVA-044 (as a thermal initiator). The original publication
listed the solution as 4% formaldehyde, 4% acrylamide,
and 0.05% bis-acrylamide. The structural rigidity andporosity of the hydrogel are a function of the concentration of these reagents. Figure 3 shows the effect ofvarying concentrations of acrylamide and bisacrylamide on tissue expansion and clearing. If the goal
is to examine an endogenous fluorescent marker, werecommend using the 4% formaldehyde and 4% acrylamide mixture to ensure the tissue structure is wellpreserved. Conversely, if the goal is to perform immunohistochemistry on the tissue, we recommend using ahydrogel composition with 3% acrylamide and 3%
formaldehyde to facilitate antibody penetration.

Figure 7 Examining effects of different microscopes to image
clear brains. Although a light sheet or confocal microscope may
be ideal, when sufficiently clear, tissue may be imaged using
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Optimal clearing time and temperature
Running the ETC at 20 V for 5 d produces optimal results
for clearing intact mouse brains. For the first 4 d, thetemperature of the clearing solution was kept at 37°C. On
the final day, the temperature is increased to 55°C tocomplete the clearing. In our experience, this pulse ofhigher temperature produces more transparent brain tissue than is produced by running the ETC at 37°C alone.
Conversely, while running ETC at 55°C for several days
produces very clear tissue, the brain tends to lose structural integrity (Fig. 3). The transparency of the tissue
increases during ETC over the course of 5 d. After 1-3 d ofclearing, the imaging depth of the brain is limited toseveral hundred micrometers, but we observed a noticeable increase in the imaging depth following 5 d of ETC at
37°C. A far greater increase in imaging depth of at least 2
mm is achieved in a period of 5 d if the final day of clearing
is conducted at a higher temperature of 55°C (Fig. 4). The
improved imaging ability following our combined 37/55°C
clearing protocol could result from either increased penetration of the propidium iodide or increased tissue transparency and deeper light penetration (although both aredesirable effects). While we cannot eliminate the possibility that the propidium iodide was better able to penetrate
the tissue following more complete clearing, we can demonstrate that our optimal clearing protocol does greatly
enhance the depth at which an endogenous fluorescentmarker can be imaged (Fig. 5). Therefore, the improved
imaging depth is primarily a result of improved transparency of the tissue.
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Figure 8 Using CLARITY to analyze neuron morphology. A, GFP-labeled cortical pyramidal neuron visualized in a CLARITYprocessed tissue section (HSV-GFP viral vector). B–E, The same neuron shown with the Z-depth limited to consecutive 40-␮m-thickhypothetical sections. Quantification of dendritic processes based on any individual 40 ␮m section will produce an incomplete
representation of a complex neuron. F, A 40-␮m-thick slice of the same neuron centered on the soma is shown. G, A tracing of theentire neuron is shown. H, The neuron is rotated around the y-axis to show how much of the dendritic arbor would be lost using
standard section thickness. The inner frame is 40 ␮m thick. I–L, Example data automatically collected using Imaris from the apicaland basal dendrites of the traced neuron. The complete neuron was compared with a single 40-␮m-thick portion of the neuron
centered on the cell body.

Glycerol versus FocusClear for refractive index
matching
After clearing, the tissue must be transferred to an imaging medium with a refractive index that matches that of
the tissue. Several options are available. FocusClear andBrainClear are commercially available solutions, which are
preferred by several groups. Another option is to use 80%
glycerol as the immersion solution. Here we compared theefficacy of glycerol with FocusClear by incubating cleared
3-mm-thick coronal brain sections in either medium. We
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observed little difference between FocusClear and 80%
glycerol other than cost (Fig. 6). Excellent quality images
can be acquired in both mediums and similar imaging
depth is achievable. Therefore we routinely use 80% glycerol as our preferred refractive index matching solution.
Optimized CLARITY can be imaged without
specialized microscopes
Our optimized CLARITY protocol produces extremely
clear tissue with preserved morphology and fluorescenceeNeuro.sfn.org

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Movie 1 A complete neuron infected with HSVGFP. The complete neuron, which is traced inred, is compared with a 50 ␮m subsection of
the neuron centered on the cell body shown inblue.

that can be used for various applications, including anatomical pathway tracking or brain-wide activity mapping.

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In principle, subsequent imaging may be conducted on avariety of different microscopes. Based on the excitation
mechanisms used by each type of microscope, we believe that light sheet microscopy is optimal for fast imaging of large tissue volume with confocal or multiphoton
imaging as slower alternatives. However, not all laboratories have access to these types of microscopes. Therefore, we tested whether wide-field epifluorescent
microscopy could be used to image CLARITY-cleared
tissue. In this study, we exposed an activity-reportermouse (ArcTRAP; Guenthner et al., 2013) to a novel context. We then perfused the mouse with a hydrogel solution, sectioned the brain into 2-mm-thick sections, andcleared the tissue. Figure 7 shows an image stack collected manually (5 ␮m z-steps) on a Nikon eclipse epifluorescent microscope equipped with a mercury arc lamp
and 10⫻ 0.3 NA air objective. An iterative, blind deconvolution algorithm was run on the resulting image stack.
The tdTomato signal can be clearly visualized through at
least 600 ␮m of the 2 mm slice. Although a more powerfulmicroscope allows higher resolution imaging, these images show that an epifluorescent microscope may proveadequate for tasks such as cell quantification in thicktissue sections. The simple addition of a motorized stage

Figure 9 Image resolution in cleared tissue. A, The USAF test target pattern imaged with a 10⫻ air objective on a Nikon lightmicroscope. The lines in element 6-6 and 7-6 are ⬃4.4 and 2.2 ␮m thick, respectively. B, The same test pattern is shown with acleared brain (⬃6 mm thick) over the pattern. The 2.2-␮m-thick lines can still be resolved through the tissue. C, Analysis of dendritic
spines is possible in cleared tissue using a confocal microscope with 25⫻ oil objective. D, Axonal filopodia in the mossy fiber pathway
can be observed in thick tissue sections.

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Movie 3 A fly-through reconstruction of thevasculature of the mouse lung after staining forcollagen.

Movie 2 An image stack showing the 3D structure of a mouse intestine labeled with propidium iodide.

with the Imaris Filament tracer module. Information regarding the 3D nature of the neuron can quickly be assessed including branching patterns, dendrite lengths,
and volumes. Figure 8, A–L (see also Movie 1), illustrates
the extent of the neuron that may be missed by standard
histology (using a 40-␮m-thick tissue section). As can beseen in this image, both the size of the dendritic arbor andthe branch order are greatly underestimated if analysis isconfined to a 40-␮m-thick substack. In addition, differ-would also allow for low-cost automated acquisition of
thick tissue sections on a very basic microscope.
Application of CLARITY for imaging neuronalmorphology
The complexity and size of many types of neurons makes
it difficult to fully analyze their morphology using standard
histology approaches. Golgi staining can be used to analyze branching and spine densities in relatively thicktissue sections, but this technique is difficult to reliably
combine with immunohistochemistry for a more detailed
analysis. Imaging of fluorescently labeled neurons allows
for combined morphological analysis with immunohistochemistry, but without tissue clearing the thickness of thesection that can be used is quite limited. As a result, theanalysis of the neuronal morphology is typically based on
only a portion of the entire neuron or complex and timeconsuming reconstructions are required. Imaging neurons
in cleared tissue is ideal as the entire neuron can bevisualized without physical reconstruction. In addition to
the information that is gained in the z-axis, CLARITY
produces images with a high signal-to-noise ratio due tothe transparent nature of the tissue. This makes CLARITY
ideal for automating the analysis of neuronal morphology
in intact tissue. We demonstrate these capabilities byimaging a complete GFP-labeled pyramidal neuron andautomatically detecting and reconstructing the neuron
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Movie 4 A 3D reconstruction showing a mouse
testis labeled with propidium iodide.

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Figure 10 Using CLARITY to clear and image other organs. A, Mouse spleen before and after CLARITY and low- and high-magnification imagesof CD34 immunohistochemistry on cleared tissue. B, Cleared mouse intestine stained with propidium iodide. C, Cleared mouse kidney labeled
for smooth muscle actin. D, Mouse testis stained with propidium iodide. E, Mouse lung immunolabeled for collagen. F, Section of mouse muscle
stained with propidium iodide. All fluorescent images were collected using a LaVision Biotec light sheet microscope except for the highmagnification image of the spleen, which was captured using a Zeiss confocal microscope.

Table 3 CLARITY troubleshooting
Problem
The tissue is
not clearing

The tissue is“melting”
The tissue isturning dark
yellow ororangeThe brain has a
black residue on
the surface

Cleared tissue isturning opaquein glycerol
An opaque precipitate
is forming in thetissue in FocusClear

May-June 2015, 2(3) e0022-15.2015

Solution
Closely monitor the pH of the clearing solution to ensure it remains between
8 and 8.5. The pH of the solution will decrease over time and solution may need
to be replaced during the clearing process. Small volume recirculators will need
to be changed more frequently than those that hold a large volume of SDS.
High temperatures for a prolonged period of time may lead to melting. Ensure thatthe clearing solution temperature is appropriate. It may be necessary to increase
the flow rate of the recirculator to maintain a suitable temperature in the chambers.
This may be due to high clearing temperature (see above). A localized burning/color
change may be the result of the tissue directly contacting an electrode. Tissue should
be in close apposition to, but not directly touching, the electrodes. A plastic mesh divider
should be placed between the tissue and electrode to prevent direct contact.
Ensure the cleanliness of the recirculator and filter. A large buildup of black residue
on the platinum electrodes can be cleaned by reversing the polarity of the electrodes andrunning the ETC chambers for several hours. Always ensure that as much hydrogel as
possible is removed from the surface of the tissue before beginning the clearing procedureas the hydrogel has a greater tendency to gather residue than the tissue itself.
On occasion, tissue may become opaque following transfer into 80% glycerol. Thiscan be corrected by returning the tissue back to PBST at 50°C until clear, typicallya few hours. The tissue can then be transferred back to glycerol. This issue seems
to be the result of incomplete tissue clearing.
A seemingly irreversible black precipitate may begin to form in the tissue if it is left
for prolonged periods of time in FocusClear. Tissue should not be stored in FocusClear any
longer than necessary to equilibrate and image the tissue.

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Figure 11 Dissection and clearing of brain subregions. As it may not be necessary to clear the entire brain, it is more efficient to dissect
the region of interest before clearing and imaging. Examples of dissected hippocampus and olfactory bulbs before and after clearing
and subsequently stained with propidium iodide are shown. Electrophoretic tissue clearing was performed for 3 d (2 d at 37°C and
1 d at 55°C). Imaging of the cleared tissue was performed with a Zeiss 710 confocal microscope and 10⫻ objective.

ences in the ratio between apical and basal dendrites may
be observed when observing the entire neuron rather thana portion of it. An important consideration may be theresolution and resulting accuracy with which neurons canbe reconstructed using CLARITY versus serial sections.
We have not performed an exhaustive comparison ofneurons imaged using CLARITY compared with thosereconstructed from serial sections. However, the resolution obtained while imaging in deep tissue appears sufficient for accurately quantifying spines and axonal
filopodia. Ultimately, the resolution that is achievable is afactor of the imaging setup; however, even with a lowcost 10⫻ air objective on an epifluorescent microscope, a
May-June 2015, 2(3) e0022-15.2015

resolution of at least 2 ␮m can be achieved at a depth of⬃6 mm (Fig. 9).
Application of CLARITY for imaging other tissue
types
CLARITY was originally described for the analysis of brain
tissue. However, other organs can also be cleared andimaged using the same protocol. The present clearing
protocol can be used to successfully clear spinal cord,
intestine (Movie 2), lung (Movie 3), spleen, intestine, testis(Movie 4), kidney, adrenal gland, skin, and muscle. Little
modification is required to clear most other organs (Fig.
10). However, some tissues, such as liver, are more diffieNeuro.sfn.org

Methods/New Tools

cult to clear with this method. If the tissue is still quite
opaque after the first 4 d of ETC at 37°C, it may benecessary to extend the time at this temperature for an
additional 1 d or 2 d before switching to 55°C. Further
troubleshooting information is provided in Table 3.

Conclusions
CLARITY, when optimized, provides a powerful method
for producing transparent and stable tissues that can bestained and imaged in their entirety. We have shown two
functional metrics that demonstrate the degree of clearingthat is accomplished with our protocol. First, we are able
to image the cleared brains using a simple epifluorescent
microscope. Second, once the tissue is adequately clear
it is possible to use glycerol as a refractive index matchingsolution rather than FocusClear.
Our methods for producing consistently clear tissue arebased on the following principles. (1) If it is not necessaryor possible to image a complete piece of tissue or brain
then it is faster and simpler to section or dissect a subregion of the tissue before the clearing process (Fig. 11).
(2) It is best to use a structurally rigid hydrogel solution
when perfusing the tissue. This ensures that the tissue will
be maximally preserved and undergo minimal transformation during the clearing, staining, and imaging steps of theprocedure. (3) To completely clear the tissues in a reasonable amount of time, active clearing with ETC is necessary. (4) Potential structural damage to the tissue canbe prevented by carefully maintaining the temperatureduring ETC, using low voltage and preventing any contact
between the tissue and electrodes. (5) To create extremely clear tissue, we find the best ETC procedure is to
run the samples at 37°C for 4 d followed by a fifth day withthe temperature increased to 55°C. One day of clearing at
55°C does not appear to have any negative effects on thetissue and has the positive effects of finishing the clearing
more effectively and also helping to reduce tissue expansion.

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