Poguzhelskaya et al. Molecular Neurodegeneration 2014, 9:19
http://www.molecularneurodegeneration.com/content/9/1/19

METHODOLOGYOpen Access

Simplified method to perform CLARITY imaging
Ekaterina Poguzhelskaya1,2†, Dmitry Artamonov1,2†, Anastasia Bolshakova1,2, Olga Vlasova1,2
and Ilya Bezprozvanny1,2,3*

Abstract
Background: Imaging methods are used widely to understand structure of brain and other biological objects.
However, sample penetration by light microscopy is limited due to light scattering by the tissue. A number ofmethods have been recently developed to solve this problem. In one approach (SeeDB) simple procedure forclarifying brain samples for imaging was described. However, this method is not compatible with immunostaining
approach as SeeDB-prepared tissue is not permeable to the antibodies. Another technique for clearing brain tissue(CLARITY) was optimized for immunochemistry, but this method technically much more demanding than SeeDB.
Results: Here we report optimized protocol for imaging of brain samples (CLARITY2). We have simplified andshortened the original protocol. Following hydrogel fixation, we cut brain tissue to 1–1.5 mm thick coronal slices.
This additional step enabled us to accelerate and simplify clearing, staining and imaging steps when comparedto the original protocol. We validated the modified protocol in imaging experiments with brains from line M Thy1-GFP
mouse and in immunostaining experiments with antibodies against postsynaptic protein PSD-95 and striatal-specificprotein DARPP32.
Conclusions: The original CLARITY protocol was optimized and simplified. Application of the modified CLARITY2 protocol
could be useful for a broad range of scientists working in neurobiology and developmental biology.
Keywords: CLARITY, See deep brain, Neuronal structure, 3D brain tissue reconstruction, Neuroimaging, Confocal,
Two-photon

Background
Understanding structural organization of the brain is
critical for modern neuroscience studies. A number ofadvanced imaging methods have been developed forstudies of brain structure. However, due to light scattering the penetration of light into brain tissue is limited.
For confocal microscopy the depth of imaging is limited
to 30 micron, for two-photon microscopy it is no morethan 600–800 microns [1]. Imaging at such depths also
require use of strong excitation power, causing local
overheating of the sample. To alleviate the problem oflight scattering, recently several procedures have beendeveloped for obtaining optically transparent brain sample for imaging. The simple and fast protocol called See
Deep Brain (SeeDB) method was initially published [2].
* Correspondence: ilya.bezprozvanny@utsouthwestern.edu†
Equal contributors
1
Department of Medical Physics, St.Petersburg State Polytechnical University,
St Petersburg 195251, Russia
2
Laboratory of Molecular Neurodegeneration, St.Petersburg State
Polytechnical University, St Petersburg 195251, Russia
Full list of author information is available at the end of the article

The SeeDB procedure takes only 1 week, requires
minimum reagents and efforts and does not transformthe sample. Prepared SeeDB samples are transparent tovisible light but not permeable for macromolecules orantibodies. Thus, SeeDB procedure is not compatible
with immunostaining. Other methods have been reported that use chemical solvents with high refractiveindex to reduce light scattering [3-5]. Similar to SeeDB,
these methods are not compatible with immunostaining
applications.
The most recently developed method is CLARITY [6].
This method is based on transforming the intact brain
tissue to the nanoporous hydrogel formed by crosslinked three-dimensional network of hydrophilic polymers. The lipid phase of the brain is solubilized indetergent and removed by electrophoresis. As a result,optically transparent brain sample is obtained for imaging studies. The nanoporous hydrogel is permeable
to the antibodies and other macromolecules, which makes
CLARITY compatible with immunostaining applications.
However, several steps of published CLARITY protocol [6]© 2014 Poguzhelskaya et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of theCreative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public
Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in thisarticle, unless otherwise stated.

Poguzhelskaya et al. Molecular Neurodegeneration 2014, 9:19
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are technically difficult. In particular, electrophoretic clearing of the sample is a time consuming and challenging stepin the CLARITY procedure.
New in method

Here we describe a simplified version of the CLARITY
procedure (CLARITY2). The initial hydrogel fixation procedure is identical to the original protocol [6]. Followinghydrogel fixation, we cut brain tissue in 1–1.5 mm thickcoronal slices using vibratome. This additional step enabled us to accelerate and simplify clearing, staining andimaging steps when compared to the original protocol.
Using CLARITY2 procedure, we have been able to obtainhigh quality confocal and two-photon images of neuronal
structures from Thy1-GFP transgenic mice [7] and following immunostaining of brain samples with antibodies
against synaptic protein PSD-95 and striatal-specific protein DARRP-32.

Results and discussion
The adult wild type mice (FVB strain) was used todevelop the protocol. The mouse brain was obtainedand embedded into hydrogel by following previously
described protocol [6] without any modifications (seeMethods for details). The next step in the publishedprotocol is clearing from lipid/detergent micelles. The
original procedure [6] requires use of electrophoretic
clearing chamber (ETC.) for the purpose of clearing thesample. This step is technically difficult and numerous variations of ETC. chamber design have been described sinceinitial publication (forum.claritytechniques.org). However,Page 2 of 5

based on our own experience and experience of other laboratories (forum.claritytechniques.org) the clearing step
remains challenging and takes substantial amount of timeand effort. We reasoned that clearing whole brain is complicated and slow process, but for most applications it is
not necessary to keep whole brain intact. Using vibratome(World Precisions Instruments) we cut the hydrogen-fixedmouse brains to 1–1.5 mm thick coronal slices (Figure 1A).
These slices were put into 50 ml conical tubes filled with
10 ml of clearing solution for 7 days (37°C, shaking at
21 rpm, fresh solution changes every 3 days). We discovered that this simple step resulted in efficient clearing of
the slices (Figure 1A). In order to speed up clearing
procedure standard protein electrophoresis chamber(for example from BioRad) can also be utilized. If electrophoresis chamber is used, then the slices clearing
procedure takes 2–3 days when compared to 7 days
with passive clearing. The quality of images obtainedwith passive and electrophoresis-based approach was
similar, and we present images from passively cleared
samples in this paper.
In our pilot experiments the cleared slices were immunostained with mouse monoclonal antibodies against
post-synaptic density protein PSD-95 and rabbit monoclonal antibodies against striatal specific protein DARPP32.
Original CLARITY procedure required 2 weeks incubation
with primary and secondary antibodies to enable sufficient
penetration of antibodies into the sample [6]. Coronal slices
in our experiments could be efficiently stained after 2 days
incubation with primary antibodies and overnight incubation with secondary antibodies. The stained structures were

Figure 1 Simplified CLARITY clearing procedure. (A) Hydrogel-embedded coronal slices before (bottom) and after (top) passive clearing
procedure. (B) Coronal slices on microscope cover glass prepared for imaging. (C) Confocal images of striatal region co-stained with DARPP32(green) and PSD-95 (red) antibodies. (D) Confocal images of hippocampus region stained with PSD-95 antibodies (red). Scale bar is 150 microns
on panels C-D.

Poguzhelskaya et al. Molecular Neurodegeneration 2014, 9:19
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visualized by Alexa-488-conjugated anti-rabbit and Alexa594-conjugated anti-mouse secondary antibodies. For
imaging experiments coronal slices were positioned on
microscope slides in PBST solution and covered by acover slip (Figure 1B). Imaging in flat slices (Figure 1B)
did not require use of FocusClear solution needed forimage reconstruction at depths more than 2 mm [6].
Using confocal microscope (Thorlabs) equipped with
20x Olympus objectives we have been able to easily
visualize the DARRP-32-positive and PSD-95-positive(Figure 1B) structures in the brain samples. The example of striatal region co-stained for PSD-95 (red)and DARPP-32 (green) is shown on Figure 1C. The
hippocampal region stained for PSD-95 (red) is shown
on Figure 1D. We attempted to compare the quality ofimages obtained with original [6] and modified procedure,
but run into technical difficulties with ETC-clearing stepwhen using original protocol (data not shown). As a result,
we have not been able to obtain cleared brain samples using
original procedure.
To demonstrate versatility of the modified method wealso processed the sections obtained from Thy-GFP mice(line M) brains [7]. In these experiments 1–1.5 mm thickcoronal sections from line M mice were passively clarified
as described above. The images of GFP-positive hippocampal neurons were collected using confocal microscope
equipped with 20x (Figure 2A) or 60x (Figure 2B) Olympus
objectives.
In the next series of experiments we used anti-PSD95
mouse monoclonal antibodies and Alexa-594-conjugatedanti-mouse secondary antibodies to stain coronal sections
from line M brains. Obtained confocal images were usedfor 3D reconstruction of neuronal structures (Figure 3A
and B). On these images individual GFP-positive neurons(green) are surrounded by PSD-95-positive synaptic structures (red).
The results shown on Figures 1, 2 and 3 validate
the modified procedure for confocal imaging andPage 3 of 5

immunostaining experiments. Using 20x objectives wehave been able to image up to depth of 250 microns
using confocal microscopy (Figure 1, 2 and 3). Whenthe samples were imaged with two-photon microscope(Thorlabs, 20x Olympus objective), the depth of penetration was increased up to 1–1.5 mm, that is spanningwhole thickness of the slice. The 3D reconstruction ofneuronal shapes based on two-photon imaging data isshown for line M GFP-positive neurons on Figure 4
and Additional file 1.
In conclusion, here we describe modified and simplified version of CLARITY protocol (CLARITY2). Following imbedding into the hydrogel, the brain samples were
cut into 1–1.5 mm thick coronal slices by vibratome. As
a result, clearing, immunostaining and imaging procedures have been accelerated and simplified. Clearing
could be performed by passive incubation of slices in theclearing solution for 1 week and did not require a special
ETC. chamber. Active clearing could be also done withbrain slices using standard protein electrophoresis chamber to shorten the clearing procedure to 2–3 days induration. Antibody staining and imaging steps were also
simplified and accelerated when compared to the original protocol. We achieved 250 micron penetration into
the thickness of the slice using confocal microscope andhave been able to image whole thickness of the slice(1.5 mm) using two-photon microscopy. Imaging in flatslices could be performed on microscope slides and did
not require use of expensive FocusClear solution needed
for image reconstruction at depths more than 2 mm [6].
We hope that described CLARITY2 procedure will be
useful for mapping neuronal circuits in mice brain andfor imaging post-mortem human brain samples. When
compared to original CLARITY procedure [6] analysisof images obtained using CLARITY2 procedure may require digital “stitching” of images from adjacent coronal
sections, but this can be done relatively easily when necessary using image analysis software.

Figure 2 Confocal images of hippocampal neurons from line M Thy1-GFP mouse. (A) Neuronal structures of hippocampus, 20x objective.
Scale bar is 300 microns. (B) 3D reconstruction of individual GFP-positive neuron, 60x objective.

Poguzhelskaya et al. Molecular Neurodegeneration 2014, 9:19
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Page 4 of 5

Figure 3 Confocal images of striatal and hippocampal neurons from line M Thy1-GFP mouse. (A, B). 3D reconstruction of GFP-positive
neurons (green) and PSD95 staining (red) is shown. 20x objective was used. Different brain regions are shown on panels (A) and (B). Scale bar is
100 microns on panels A and B.

Methods
Mice

Wild type FVB/NJ (Stock Number: 001800) or line M
mice Tg(Thy1-EGFP)MJrs/J (Stock Number: 007788) [7]were used. Both lines were obtained from the JacksonLaboratory and the breeding colonies were establishedin the animal facility located in the Laboratory of
Molecular Neurodegeneration in St Petersburg State
Polytechnical University. All mouse experiments havebeen performed according to the procedures approvedby the local animal control authorities.
Hydrogel and clearing solutions

Both solutions were prepared by following protocol described in the original article [6]. Briefly, to preparehydrogel solution combine and mix 40 ml of acrylamide(40%), 10 ml of bis-acrylamide (2%), 1 g of VA-044 initiator (10% wt), 40 ml of 310 PBS, 100 ml of 16% PFA and
210 ml of dH2O. Hydrogel solution is stored at −20°C, allmanipulations with hydrogel are performed on ice. For
clearing solution combine and mix 123.66 g boric acid,
400 g sodium dodecyl sulphate, and 9 l dH2O. Add
dH2O to 10 l and add NaOH until the pH has reached8.5. This solution can be made, stored and used at room

temperature (20°C). To avoid skin contact or inhalation,
prepare solutions in a fume hood wearing gloves while
preparing both of them.
Mice perfusion

Thaw the 40 ml of hydrogel at room temperature.
Gently mix the content, avoid air bubbles. Keep the tube
with hydrogel on ice. Provide animal anesthesia – in our
experiments mice were anesthetized with 300 ul of
Urethane (250 mg/ml). Transcardially perfuse the mousewith 20 ml of ice-cold solution of hydrogel with perfusion rate of 10 ml per minute. Quickly extract the brain
and place it in a conical tube containing remaining
20 ml of hydrogel. Wrap tube in aluminum foil if thesample contains fluorescent substance (such as GFP) to
avoid photobleaching. Place the tube in a refrigerator(4°C) for 1–2 days to ensure full penetration of the sample by hydrogel.
Hydrogel tissue embedding

Tissue embedding was performed according to theprotocol described in the original article [6]. Briefly,
50 mL conical tube containing hydrogel-soaked brain
sample was positioned to the dessication chamber in a

Figure 4 Two-photon imaging of hippocampal neurons from line M Thy1-GFP mouse. Single frame from 3D neuronal image reconstruction(Additional file 1). 20x objective was used. Scale bar is 150 micron.

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fume hood. The dessication chamber is filled with the nitrogen gas from the tank. Switch the desiccation chamber
valve from nitrogen gas flow to the vacuum pump. Keepthe vacuum on for 10 minutes, then switch the vacuum off
and slowly fill the dessication chamber with the nitrogengas. Open the dessication chamber and tightly close the topof the sample tube. Minimize exposure of the sample to airas oxygen impedes formation of hydrogel. Submerge thetube in 37°C incubator on the rotator. Incubate for 3 hours
or until solution has polymerized. In a fume hood, extract
the hydrogel-embedded brain sample from the gel.

Confocal and 2 –photon imaging systems (ThorLabs)
equipped with 20× and 60× Olympus objectives (w.d.
2.0 mm) were used in our imaging experiments. Obtained
images were analyzed using Imaris v7.4.2. Software.

Tissue clearing

Authors’ contributions
EP and DA performed the experiments. EP, DA, AB analyzed data. EP, DA,
OV and IB designed research. EP, DA, AB, OV, IB wrote the paper and prepare
figures for publication. All authors read and approved the final manuscript.

Coronal slices (1–1.5 mm in thickness) were cut fromhydrogel-embedded brain using World Precisions Instruments vibratome (NVSLM1 Motorized Advance
Vibroslice). The slices are numbered, placed in 50 ml
plastic tubes and incubated in 10 ml clearing solution
for 7–10 days at a temperature of 37C with shaking
21 rpm. Fresh clearing solution was exchanged every
3 days. The clearing of tissue was evaluated by eye. In
most experiments 7 days was enough for complete clearing,
but for some samples additional 3 days of clearing wasneeded. The standard Bio-Rad chamber for Western blotting (Mini-Protean Tetra Cell system # 165–8000) could be
used to speed up a clearing to 2–3 days in needed. Ifelectrophoretic clearing is used, the chamber is placed to athermostat at 37°C, and the voltage is set for 12 V.
Following passive (7 days) or electrophoretic (2–3days) clearing the slices are washed from clearing solution in PBST (0.1% Triton X- 100 in PBS) for 2 days.
The slices are now clarified.
Immunochemistry

The cleared brain slices are placed in PBST. The primary
antibody is added at 1:1000 dilution for 2 days, then slices
are washed with PBST 5 times for 30 minutes. The secondary antibody is added at 1:1000 overnight in PBST, and theslices washed once for 30 min in PBST. The antibodies
used in our experiments were: mouse monoclonalanti-PSD-95 (Pierce, MA1-045), rabbit monoclonalanti-DARPP32 (Cell Signaling 19A3), Alexa Fluor488 Goat
anti-rabbit (Life Technologies, A-11008), Alexa Fluor594
Goat anti-mouse (Life Technologies, A-11005).
Imaging

The slices are removed from PBST and put on a glass
slide. The slices are covered with the cover glass, whichis cemented to the glass slide using plastiline (Figure 1B).
The coronal slices are put on the microscope slide in thesmall drop of liquid (PBST) to avoid drying and to prevent formation of air bubbles. The plastiline is mounted
around the sample and the sample is covered with acover slip. Be very careful to avoid trapped air bubbles.

Additional file
Additional file 1: 3D reconstruction of hippocampal neurons form line
M Thy1-GFP mice based on two-photon imaging data (20 x objective).
Competing interests
The authors declare that they have no competing interests.

Authors’ information
Ekaterina Poguzhelskaya at poguzhelskaya@mail.ru is a contact for technicalquestions on the procedures described in this paper.
Acknowledgements
We would like to thank the members of Laboratory of Molecular
Neurodegeneration in St Petersburg State Polytechnical University for their
help and support in the course of these studies. This work was supported by
the contract with the Russian Ministry of Science 11.G34.31.0056 (I.B) and NIH
grants R01NS074376 and R01NS080152 (I.B.).
Author details
1
Department of Medical Physics, St.Petersburg State Polytechnical University,
St Petersburg 195251, Russia. 2Laboratory of Molecular Neurodegeneration,
St.Petersburg State Polytechnical University, St Petersburg 195251, Russia.
3
Department of Physiology, University of Texas Southwestern Medical Center
at Dallas, Dallas, TX, USA.
Received: 27 March 2014 Accepted: 20 May 2014
Published: 26 May 2014
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Cite this article as: Poguzhelskaya et al.: Simplified method to perform
CLARITY imaging. Molecular Neurodegeneration 2014 9:19.