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Chemical Principles in Tissue
Clearing and Staining Protocols
for Whole-Body Cell Profiling
Kazuki Tainaka,1 Akihiro Kuno,2,3 Shimpei I. Kubota,1Tatzya Murakami,1 and Hiroki R. Ueda1,4
1

Department of Systems Pharmacology, The University of Tokyo, Tokyo 113-0033, Japan2

Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba,
Ibaraki 305-8575, Japan

3

PhD Program in Human Biology, School of Integrative and Global Majors, University ofTsukuba, Ibaraki 305-8575, Japan

4

Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, Suita,
Osaka 565-0871, Japan; email: uedah-tky@umin.ac.jp

Annu. Rev. Cell Dev. Biol. 2016. 32:713–41

Keywords

First published online as a Review in Advance onJune 8, 2016

tissue clearing, fixation, permeabilization, decolorizing, RI matching,chemical staining

The Annual Review of Cell and Developmental
Biology is online at cellbio.annualreviews.org
This article’s doi:
10.1146/annurev-cellbio-111315-125001
c 2016 by Annual Reviews.
Copyright All rights reserved

Abstract
Mammalian bodies have more than a billion cells per cubic centimeter, whichmakes whole-body cell (WBC) profiling of an organism one of the ultimate
challenges in biology and medicine. Recent advances in tissue-clearing technology have enabled rapid and comprehensive cellular analyses in wholeorgans and in the whole body by a combination of state-of-the-art technologies of optical imaging and image informatics. In this review, we focus
mainly on the chemical principles in currently available techniques for tissue clearing and staining to facilitate our understanding of their underlying
mechanisms. Tissue clearing is usually conducted by the following steps: (a)fixation, (b) permeabilization, (c) decolorizing, and (d ) refractive index (RI)
matching. To phenotype individual cells after tissue clearing, it is important to visualize genetically encoded fluorescent reporters and/or to stain
tissues with fluorescent dyes, fluorescent labeled antibodies, or nucleic acidprobes. Although some technical challenges remain, the chemical principles in tissue clearing and staining for WBC profiling will enable various
applications, such as identifying cellular circuits across multiple organs andmeasuring their dynamics in stochastic and proliferative cellular processes,
for example, autoimmune and malignant neoplastic diseases.

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Contents

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INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 714
STRATEGY FOR TISSUE CLEARING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 719
FIXATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 721
Paraformaldehyde . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 722
Hydrogel Embedding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 722
SWITCH-Based Glutaraldehyde . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 723
PERMEABILIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 723
Water-Miscible Polar Solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 724
Hyperhydration Reagents without Delipidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 725
Delipidation Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 726
DECOLORIZING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 728
REFRACTIVE INDEX MATCHING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 729
PRESERVATION OF FLUORESCENT SIGNALS FROMREPORTER PROTEINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 730
STAINING OF ENDOGENOUS COMPONENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 731
iDISCO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 733
CUBIC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 734
ScaleS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 734
CLARITY-Related Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 735
SWITCH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 735
OUTLOOK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 736

INTRODUCTION
An animal’s body has an enormous number of cells that compose various functional and structuralunits. Different cell types play crucial roles in diverse physiological systems, including the
respiratory and circulatory systems, nervous and skeletal/muscular systems, endocrine system,
digestive system, and immune system. Comprehensive analysis of each system or organ at
single-cell resolution in the body has been one of the most fundamental challenges in biology
and medicine. Remarkable progress in imaging technology such as computed tomography (CT)
(Bouxsein et al. 2010), magnetic resonance imaging (MRI) (Ogawa et al. 1990), positron emission
tomography (PET) (Shokeen & Anderson 2009), and near infrared imaging (Leblond et al. 2010)
has revealed detailed anatomical structures of living animals. However, it is still difficult to use
these methods to extract various properties of individual cells due to the lack of high-contrast
cell-labeling tools. Although conventional histology techniques are principally capable of global
phenotyping of cells in fixed tissues (Isosaka et al. 2015, Oh et al. 2014, Ragan et al. 2012),
these techniques are laborious and require tissue sectioning, which are challenging barriers to
rapid three-dimensional (3D) visualization of organ structures. By contrast, optical projection
tomography (Ntziachristos 2010, Sharpe 2004) and optical sectioning with light-sheet microscopy(Dodt et al. 2007, Keller et al. 2015) in combination with recent advances in tissue-clearing
techniques (Susaki & Ueda 2016) are promising strategies toward visualizing single cells withina fixed whole-organ or whole-body context. Continuously updated informatics tools enable hugeamounts of imaging data to be processed (Amat et al. 2015) and facilitate automated comparative
analysis (Susaki et al. 2014, 2015), imaging processing (Chhetri et al. 2015), and information
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extraction (Amat et al. 2014). Maturation of tissue clearing, rapid 3D imaging microscopy, andimage informatics could provide novel biological and medical platforms for whole-body cell(WBC) profiling, allowing us to see both the forest and the trees (Economo et al. 2016, Keller& Ahrens 2015, Ode & Ueda 2015, Oh et al. 2014, Ragan et al. 2012).
Tissue clearing is the first critical step toward WBC profiling. A number of tissue-clearing
reagents and protocols have been developed, as shown in Table 1 and Figure 1. The pioneering
work by Werner Spalteholz first introduced the principle of transparent 3D specimens a century
ago (Spalteholz 1914). Dodt and colleagues built upon Spalteholz’s achievements by applying abenzyl alcohol/benzyl benzoate (BABB)-based clearing protocol to their original ultramicroscopy,
which enabled rapid 3D imaging of whole organs based on light-sheet fluorescence microscopy(LSFM) (Dodt et al. 2007). Subsequently, Dodt and colleagues explored GFP-friendly organic
solvents from a large library of more than 10,000 chemicals and discovered highly effective onesin tetrahydrofuran (THF) and dibenzylether (DBE) (Becker et al. 2012). These protocols are
summarized as 3DISCO (Ertürk et al. 2012). The Tessier-Lavigne group has established a rapid
whole-mount staining protocol termed iDISCO, which is based on 3DISCO (Renier et al. 2014). In
addition to conventional hydrophobic organic chemical cocktails, hydrophilic reagents—including
sugars (e.g., glucose), alcohols (e.g., glycerol, polyethylene glycol, butanediol, trimethylolpropane,sorbitol, xylitol), dimethyl sulfoxide (DMSO), and oreic acid—have been used (Susaki & Ueda
2016, Tuchin 2015). Recently, the Imai group developed a clearing method termed SeeDB, which
is based on fructose (Ke et al. 2013). The Miyawaki group used hyperhydration urea (Hama et al.
2011), which led to other urea-based clearing protocols, including CUBIC (Susaki et al. 2014),
FRUIT (Hou et al. 2015), ClearSee (Kurihara et al. 2015), and ScaleS (Hama et al. 2015). ScaleS,
which uses both urea and sorbitol, has improved clearing performance without disturbance of intact cell structures. In contrast to ScaleS, CUBIC sought to promote more effective delipidation toensure sufficient clearance of specimens so that whole-organ images at single-cell resolution could
be acquired by LSFM. Aminoalcohols in the CUBIC cocktail had an unexpected decolorizing ability, resulting in clearance of blood-infused tissue in addition to efficient lipid solubility (Tainaka
et al. 2014) and thus enabling 3D anatomy and pathology of various organs at single-cell resolution by rapid whole-body and whole-organ imaging. The Deisseroth group developed CLARITY,
which is a combination of hydrogel-embedding fixation and electrophoretic delipidation (Chung& Deisseroth 2013, Chung et al. 2013). Endogenous proteins were well preserved by covalentcross-linking in an acrylamide gel. Delipidation by the ionic detergent was dramatically accelerated by the electric field potential. More recently, CLARITY was improved by various related
techniques, including PACT-PARS (Yang et al. 2014), stochastic electrotransport–based clearing (Kim et al. 2015), CLARITY-TDE (Costantini et al. 2015), and ACT-PRESTO (Lee et al.
2016). The Gradinaru group has also greatly advanced these detergent-based clearing protocols
and has provided many fruitful applications, including optimization of hydrogel-based fixation,
passive delipidation (PACT), perfusion-based delipidation (PARS), highly effective RI adjustment (RIMS), decalcification (PACT-deCAL), and tissue clearing through expansion (ePACT)(Treweek et al. 2015, Yang et al. 2014). The Chung group has strongly contributed to versatilestaining principles based on stochastic electrotransport and binding-controlled passive diffusion
in a development termed SWITCH, which is backed by well-founded theory (Kim et al. 2015,
Murray et al. 2015).
All current techniques result in tissue clearing by similar physical principles, despite different
chemical approaches. However, as far as we know, the underlying chemical principles have notbeen highlighted. In this review, we mainly discuss the chemical principles for major clearing
protocols and introduce emerging chemical staining methods.

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PFA

ClearT , ClearT2

Scale

FRUIT

ScaleS

SeeDB

SeeDB2
CUBIC

Kuwajima et al. 2013

Hama et al. 2011

Hou et al. 2015

Hama et al. 2015

Ke et al. 2013

Ke et al. 2016

Susaki et al. 2014,2015; Tainaka et al.
2014

PFA

PFA

PFA

PFA

PFA

ScaleCUBIC-1(urea/aminoalcohol/Triton
X-100): Urea: hydration and molecular
influx Aminoalcohol/Triton X-100:
delipidation

Saponin: delipidation

Fructose: osmotically balanced
molecular flux

ScaleS (urea/sorbitol/DMSO): Urea: hydration and molecular
influx Sorbitol: osmotically balanced
molecular flux

FRUIT (urea/fructose) Urea: hydration and molecular
influx Fructose: osmotically balanced
molecular flux

ScaleA2 (urea/glycerol/Triton
X-100) Urea: hydration and molecular
influxFormamide: dehydration orformamide-induced molecular
influx

TDE: dehydration

THF: dehydration and delipidation;
CH2 Cl2 : delipidation

EtOH, 1-PrOH, or t-BuOH:
dehydration; CH2 Cl2 : delipidation

Permeabilization

Aminoalcohol:
heme

None

Thioglycerol:
Maillard
reaction

None

Thioglycerol:
Maillard
reaction

None

None

None

H2 O2 : heme
and otherchromophores

None

Decolorizing

1.49

1.52

HistodenzTM

1.44

1.46–1.50

1.38

1.45

1.49

ScaleCUBIC-2 (urea/
aminoalcohol/
sucrose)RI

1.48–1.52

1.56

1.56

Fructose

ScaleS4(urea/sorbitol/DMSO)FRUITScaleA2

Formamide/
polyethylene glycol

TDE

DBE

BABB

RI matchHigh (3–4)

High (2–3)

Moderate (1)High (2–3)

Moderate (1–2)

Moderate (1)Moderate (1)Moderate (1)Highest (5)High (3–4)

Clearing
performance

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PFA

PFA

TDE

Aoyagi et al. 2015,
Costantini et al. 2015

PFA

PFA

3DISCO,iDISCOBABBFixation

Becker et al. 2012,
Ertürk et al. 2012,
Renier et al. 2014

Dodt et al. 2007,
Schwarz et al. 2015

Protocol

Tissue-clearing protocols for mammalian samples

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Reference

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717

Stochastic electrotransport
Expansion
microscopy

SWITCHKim et al. 2015

Chen et al. 2015

Murray et al. 2015

Glutaraldehyde(1) PFA, (2)acrylamide/bis-acrylamide/
sodium acrylate

PFA/acrylamide/bis-acrylamide

PFA/acrylamide/bis-acrylamide

Thermal clearing
SDS: delipidation

Protease: fiber digestion

ETC-based clearing
SDS: delipidation

ETC-based clearing
SDS: delipidation

SDS: delipidation
Collagenase: fiber digestion

Passive clearing (PACT) or
perfusion-based clearing(PARS)
SDS: delipidation

Thioglycerol
or sodiumsulfite:Maillard
reaction

None

SDS: heme

SDS: heme

None

SDS: heme;
Sudan Black
B: lipofuscin
autofluorescence

SDS: heme

Aminoalcohol:
heme

Aminoalcohol:
heme

Diatrizoic acid/N-methylglucamine/
iodixanol

Deionized water

Diatrizoic acid/N-methylglucamine/sorbitol

TDE

Deionized water

1.47

1.33

1.46

1.45

1.33

RIMS:
1.46–1.49;
sRIMS:
1.44

1.45

FocusClearTM ; glycerol;
RI 1.454 category
1806Y (Cargille Labs)
RIMS: HistodenzTM ;sRIMS: sorbitol

1.47

1.49

DMSO/sorbitol

ScaleCUBIC-2 (urea/
aminoalcohol/sucrose)

Highest (5)Highest (5)Highest (4–5)

PACT: high (3);
PARS: highest(3–5)

Passive clearing:high (3–4);
ETC: highest(4–5)

High (3–4)

High (3-4)

Abbreviations: BABB, benzyl alcohol/benzyl benzoate; DBE, dibenzylether; DMSO, dimethyl sulfoxide; ETC, electrophoretic tissue clearing; PFA, paraformaldehyde; RI, refractive index; SDS, sodium dodecyl sulfate;
TDE, 2,2 -thiodiethanol; THF, tetrahydrofuran.

CLARITYTDE(1) PFA, (2)acrylamide/bis-acrylamide/
sodium acrylateePACT

Costantini et al. 2015

PFA, acrylamide

PACT-PARS

Passive clearing or ETC-based
clearing
SDS: delipidation

ScaleCUBIC-1(urea/aminoalcohol/Triton
X-100): Urea: hydration andmolecular influx
 Aminoalcohol/Triton
X-100: delipidation

ScaleCUBIC-1A(urea/aminoalcohol/Triton
X-100/NaCl): Urea/NaCl: osmoticallybalanced molecular flux
 Aminoalcohol/Triton X-100:
delipidation

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Treweek et al. 2015,
Yang et al. 2014

PFA/acrylamide/bis-acrylamide

CLARITY

Chung et al. 2013,
Tomer et al. 2014

PFA

PFA

CUBIC

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Economo et al. 2016

Susaki & Ueda 2016;
http://cubic.riken.jp

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a Organic solvent–based clearing
Lipid

Fixed tissue

Dehydration, delipidation,
and/or bleaching

Protein

RI match

Water
Dehydration (water-miscible polar solvent)
OH

OH

OH
OH
TDE

t-BuOH(BABB)

1-PrOH(BABB)EtOH(BABB)

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OH

Bleaching

Delipidation

O

S

H2O2
(iDISCO)THF(3DISCO)RI match

O

OH

Cl

Cl

H H
DCM
(3DISCO)OOOHBABB, RI = 1.56

DBE, RI = 1.56
(3DISCO)S

OH

TDE, RI = 1.49

b Hydrophilic reagent–based clearing
Delipidation and/or
decoloring

Fixed tissue

Hydration

Delipidation

H2N NH2
Urea(Scale series)

Heme

O )H
n

HO
N
N
OH

HO
Triton X-100(Scale series)

O
H

DecoloringO(O

RI matchOH

Quadrol(ScaleCUBIC-1)

OH

O–)5 O S OO(SDS(CLARITY series)

N

NH2

NH

N N

NH
ScaleCUBIC-1
Sudan Black B (PACT)HOHON N OHOH
O( O )Hn

Maillard reaction
O

HS

OH
OH
Thioglycerol(SeeDB, SWITCH)

H2N NH2

Na2SO3(SWITCH)

+
+
+
+

–
O
–
( ) 5 O S O––O–Electric field potential

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OH

O

Urea-induced molecular influx and delipidation
CLARITY-related methods

O I

PARS

OH

HO

HO
O
N

OH
I O

OH

I H OHOH H I
N
N
OH
HO
O I O
HistodenzTM(RIMS, SeeDB2)

N
N
H I H
Diatrizoic acid
(FocusClearTM)

SWITCH
80°C

O( )5 O S O–OTranscardial perfusion

DMSO(ScaleS)

OH OH
OH OH
Fructose
Sorbitol(sRIMS, ScaleS)(SeeDB, FRUIT)
CH2OH CH2OH
OH
O
OH
HO
O
OH
CH2OH
OH
OH
Sucrose (ScaleCUBIC-2)
Contrast reagents, RI = 1.46–1.49

Lipofuscin autofluorescence
N N

O
S

OH OH

OH O
HO

HO
OH
Triethanolamine(ScaleCUBIC-2)

Formamide(ClearT)

RI match
HO
OH
S
OH
OH
OH
Glycerol
TDE(CLARITY)(CLARITY-TDE)
Sugars, RI = 1.44–1.49

OH
H

HO

O
( )5 O S O–
O
Thermal energy

HO

OH H I
N
O

O

O
I
N

I

OH

H
N

OH
I H
N

N
OH
I O
O O
Iodixanol (SWITCH)
I

OH
OH

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STRATEGY FOR TISSUE CLEARINGThe opacity of biological tissues is derived from heterogeneous components with different opticalproperties such as refractive index (RI) and light absorption (Tuchin 2015, Tuchin et al. 1997).
Most biological tissues are composed of 70–80% water with low RI (n = 1.33), ∼10% proteins
with high RI (n > 1.44), and ∼10% lipids with high RI (n > 1.45) ( Johnsen & Widder 1999,
Tuchin 2015). Incoming light is scattered by the heterogeneity in the RIs of these components.
In addition, light transmission decays by light absorption from endogenous pigments such asheme, riboflavin, melanin, and lipofuscin (Horecker 1943, Tuchin 2015, Weissleder 2001). In
this review, we focus on the chemical principles of tissue clearing: how various chemicals can
reduce both light scattering and light absorption. For more detailed discussion of the underlying
physics of tissue clearing, the reader is referred to reviews by Johnsen & Widder (1999) and Tuchin(2015). Tissue-clearing protocols should achieve homogenization of RI inside tissues and removalof endogenous absorbents without loss or disruption of molecules of interest. Tissue clearing is
usually conducted by the following steps: (a) tissue fixation, (b) permeabilization, (c) decolorizing,and (d ) RI matching by high-RI medium (Figure 1). Most currently available clearing protocols
have been developed to visualize specific proteins inside tissues via encoding of fluorescent reporter
proteins or postlabeling by fluorescently tagged antibodies.
In the first step, researchers should consider tissue fixation methods suitable for protein retention through the tissue-clearing protocol (Figure 2). If more efficient tissue fixation conditions arechosen to retain proteins more rigidly, tissue-clearing conditions must become harsher. Because
water molecules occupy 70–80% of the volume of tissues and exhibit much lower RI than doproteins, water inside tissues has to be substituted with a high-RI medium in the homogenization
process of RI (Liu et al. 1996, Tuchin 2015, Tuchin et al. 1997). Lipids also cause light scattering
inside tissue due to high RI. In addition, lipids, which are the major component of the plasma membrane, obstruct external medium from cells. Therefore, delipidation is also an important process
to permeabilize cell membranes and therefore render full clearing of tissues so that whole-organ
samples can be visualized by LSFM with single-cell resolution. Small pigments such as heme canbe eliminated from tissues by several clearing conditions (Lee et al. 2014, Tainaka et al. 2014,
Yang et al. 2014). Although it is still hard to remove large pigments such as melanin and lipofuscin,some chemical treatments may suppress autofluorescence from lipofuscin in elderly mammalianbrain samples (Treweek et al. 2015). Additionally, several chemical treatments, such as those by
the Maillard reaction, result in undesirable coloring. Reductive chemical treatment partially solves
these problems (Ke et al. 2013, Murray et al. 2015).
Chemically speaking, current tissue-clearing protocols are divided into two groups: organic
solvent–based clearing methods and hydrophilic reagent–based clearing methods (Figure 1). In
the former, dehydration and permeabilization occur through alcohol or ether treatment (Dodt←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
Figure 1
Overview of tissue-clearing methods. (a) Organic solvent–based clearing methods include dehydration, delipidation, bleaching, andrefractive index (RI) matching. Water-miscible polar solvents dehydrate tissue samples. Delipidation by THF or DCM contributes toclearing efficiency after RI matching. Although the water-free clearing method results in some tissue shrinkage and quenching offluorescent proteins, these protocols are simple and fast and have high clearing performance. (b) Hydrophilic reagent–based clearing
methods include delipidation, decolorizing, and RI matching. Detergents and aminoalcohols remove lipids from tissue samples.
Delipidation is facilitated by chemically or physically induced molecular flux. Urea promotes the influx of aminoalcohol and detergent
in the CUBIC protocol. Permeation of the ionic detergent SDS is driven by electric field potential (CLARITY), transcardial perfusion(PARS), or thermal energy (SWITCH). Highly water-soluble molecules such as sugars and contrast reagents are used as RI matching
medium.
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et al. 2007, Ertürk et al. 2012). Those solvents are miscible in organic solvent, and thus high-RI
aromatic solvents (n ∼ 1.56) homogeneously fill the tissue. This approach has an advantage inclearing kinetics probably due to the effective diffusion coefficient of small molecules. In contrast,
organic solvents are often harmful and attenuate fluorescent reporters. Although pretreatment ofbasic solution alleviates some quenching of fluorescent proteins (Schwarz et al. 2015), harmful

a PFA fixation
Heat, OH– HO

H H
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OHn
Paraformaldehyden = 2–8
O

O

OH

H

H

H

Monomericformaldehyde

HO

OH

H

H

LysineH

Dehydrated form

H
N

NH2

N terminus
R H
N
H2N
O

OH

H H
Methylol adductProteinb Hydrogel embedding
H H
Formaldehyde

Protein,otheramines

N

H H

R

O

Hydrogel in CLARITYO HH ON N
H H
Bisacrylamide

OH

HO
NH2

H
H H
N
N

H
N

H
N

H H O

O

N

NH2
Acrylamide

O

CH2

H

N
H H

N

N N

O HN

HN

O

HN

N
H

N
H
Thermal initiator VA-044

H2N

O

Hydrogel in PARS
H
H

NH
H
N

H2N
OH

N
H

NH2

N
N

O

N

H H

H H

O H 2N

H2N

NH2
Acrylamide

H
H

NH2

+

Methylol
adduct

N
N N
N
N
H
H
Thermal initiator VA-044

H2 N

NH2

N
N

N

PBS wash

O

N

H H

H2N

O H2N

O

N
H

N
H

O

c Glutaraldehyde fixation
pH < 7
O

pH ~ 3O

HO

Dehydrated form

O

OH

NH2

Cyclic hemiacetal form
Highly permeable
SWITCH OFF

pH ~ 7

N
H

O

N
H

Michael adduct
O
O

O

n
α, β-Unsaturated oligomeric aldehydes
Less permeable
SWITCH ON

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O

N

O

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pH ~ 7

O

O

O

NH2
Schiff base

x

y
NH

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waste, especially explosive THF and DBE, remain a problem. In the latter hydrophilic method,
fully cleared tissue samples are prepared by detergent-based delipidation and by RI matching witha concentrated sugar- or contrast agent–based aqueous medium with high RI (n ∼ 1.44–1.49).
This approach is appropriate for visualization of endogenous fluorescent proteins and is relatively
harmless in spite of redundant and laborious protocols. All current clearing protocols have bothadvantages and disadvantages, so users should determine the protocol best suited for their purpose.

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FIXATION
Tissue fixation is a fundamental step to preserve molecules of interest during the tissue-clearing
process. Most clearing protocols aim for visualization of intracellular proteins, and therefore conventional fixation protocols were based on protein-specific chemical reactions, except for several
coagulant fixations such as trichloroacetic acid (Hirashima & Adachi 2015). Recently, hydrogelbased and glutaraldehyde-based fixation methods have succeeded in the preservation of lipids andeven small molecules such as GABA and dopamine (Chung et al. 2013, Murray et al. 2015). The
Deisseroth group has revealed that postfixation of hydrogel-emmbedded tissues by 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC) significantly enhanced preservation of transcriptional
products by cross-linking of the 5 -phosphate group of RNA molecules (Sylwestrak et al. 2016).
The Briggman group has demonstrated that an osmolarity-balanced fixative buffer contributes topreservation of extracellular space (Pallotto et al. 2015). Before describing representative fixation
chemistry, we summarize key considerations for the relationship between fixation and clearing asfollows.
1. Basic amino groups such as lysine are largely consumed (depending on the degree of fixation)by the cross-link reaction during all chemical fixation processes. Thus, isoelectric points of
biomolecules inside fixed tissues are shifted in the acidic direction. Because biomolecules
such as proteins, lipids, and nucleic acids tend to be insoluble in acidic conditions, chemicalclearing is more favorable in neutral to basic conditions.
2. More highly fixed tissues require harsher clearing conditions to be fully transparentized.
Thus, researchers should choose a chemical fixation depending on the research purpose
and the available equipment. For example, to visualize signals from relatively abundant
fluorescent proteins in mice organs without specific devices such as an electrophoretic tissueclearing chamber, paraformaldehyde (PFA) or PACT fixation is more feasible because thisprotocol clears tissue by simple immersion. In contrast, SWITCH-based glutaraldehyde
fixation is more desirable for minimizing protein loss to detect tiny amounts of proteins inhuman organs by immunohistochemistry.
3. Generally, chemical fixation induces artificial and multiple chemical reactions in biologicalsystems. Architectures in the living organism are altered not only by the clearing process
but also by the initial fixation process.
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
Figure 2
Fixation chemistry of paraformaldehyde (PFA), hydrogel embedding, and SWITCH-based glutaraldehyde. Illustrated are plausible
mechanisms in covalent cross-link reaction. (a) In PFA fixation, monomeric formaldehyde prepared from polymerized PFA can activate
basic amino acids of proteins to yield methylol adducts, which then condense with other functional groups such as N-terminal aminogroups. (b) CLARITY hydrogels can form covalent cross-links between polymerized protein networks and acrylamide gel matrices,whereas these are independent in PARS hydrogels. (c) In glutaraldehyde chemistry, highly permeable cyclic hemiacetal is predominant
at approximately pH 3 and is inert to acidic polymerization (SWITCH OFF). At approximately pH 7, glutaraldehyde is polymerized toform less permeable α,β-unsaturated oligomeric aldehydes (SWITCH ON). Oligomeric aldehydes react with proteins via Schiff baseand Michael-type reactions.
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Paraformaldehyde

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PFA is one of the most widely used fixative reagents. Because monomeric PFA is a tiny molecule,
PFA is highly permeable in tissues. In the initial step, aldehyde rapidly reacts with basic aminogroups such as lysine and N-terminal amino groups of endogenous proteins to form methylol
adducts (Metz et al. 2004, 2006) (Figure 2). This step is reversible and considered to be completely
finished within 24 h at 4◦ C (Helander 1994). Thus, methylol adducts can be restored to the original
amino groups and free aldehyde by washing with phosphate-buffered saline (PBS). In the second
step, the methylol adducts are irreversibly conjugated with adjacent arginine, histidine, tyrosine,
tryptophan, asparagine, glutamine, and N-terminal amino groups of proteins. Cross-linking between arginine and tyrosine, in the Mannich reaction, can be recovered by several antigen retrieval
conditions (Sompuram et al. 2004). This latter cross-link reaction is relatively slow and may not
be completely finished in the short postfixation period in current clearing protocols. Because PFA
fixation is insensitive to lipids, lipids are efficiently washed out by detergent-based delipidation. In
addition, PFA forms only physically contiguous protein networks and fails to covalently fix spatially isolated proteins. To observe tiny amounts of proteins, researchers should use other fixative
methods that more effectively preserve protein. However, PFA fixation accelerates tissue clearing
under relatively mild conditions and is therefore suitable for preparing fully cleared specimensthat retain signal from fluorescent proteins.

Hydrogel Embedding
The Deisseroth group developed a novel fixation method by hydrogel embedding, which improved the preservation efficiency of proteins by covalent conjugation with an acrylamide gelmatrix (Chung et al. 2013). Initially, anesthetized mice or other mammalian organisms are perfused with a chemical cocktail of PFA, an acrylamide monomer (a mixture of acrylamide andN,N  -methylene bisacrylamide), and a thermal polymerization initiator. Methylol adducts between amino groups of proteins and aldehyde form after 1 day after fixation at 4◦ C (Figure 2).
During the initiation of polymerization at 37◦ C, the embedded acrylamide polymerizes in a gel
matrix, and methylol adducts are covalently attached to the amide group of acrylamide. Unlike
PFA fixation, the hydrogel embedding fixation preserves spatially isolated proteins in the gel matrix and enhances the physical and chemical strength of the specimens. However, because thisfixation also noncovalently retains lipids in the gel matrix, it is necessary to apply an electric fieldpotential in the presence of the strong detergent SDS to remove lipids. The Gradinaru group
investigated milder clearing conditions to clear the whole mouse body, including fragile tissues,and found the minimal fixation suitable for SDS-based passive clearing (Treweek et al. 2015, Yanget al. 2014). As a result, this group demonstrated that SDS-based passive clearing is compatible
with effective protein preservation by only a 4% acrylamide-based fixative without bisacrylamide
as a cross-linker. We noted that SEM images showed that pore sizes of 4% acrylamide matrices
were larger than those of 4% PFA-acrylamide matrices (Yang et al. 2014). Therefore, antibodies
more deeply penetrated into 4% acrylamide matrices than into 4% PFA-acrylamide matrices.
In this fixation, the weight and volume of tissue-hydrogel matrices after SDS-based delipidation swelled approximately twofold. But those specimens shrank to their original sizes without
significant anatomical distortion after RIMS treatment as an RI-matching step.
The Boyden group devised an alternative hydrogel-embedding method for linear tissue expansion that led to an increase in optical resolution: expansion microscopy (EM) (Chen et al.
2015). Thin brain slices were embedded into a hydrogel composed of sodium acrylate, which is awell-known water absorptive polymer, and acrylamide. Those slices underwent a 4.5-fold linear

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expansion without distortion after proteolytic treatment and deionization with distilled water. We
noted that intensive tissue swelling by water is accompanied by the reduction of RI inside tissues(Chen et al. 2015). Thus, this approach is another strategy for tissue clearing without high-RI homogenization. EM requires complicated labeling methods to visualize target proteins due to theproteolysis treatment. To address this issue, the Gradinaru group treated acrylate/acrylamideembedded rodent brain slices with collagenase in place of protease in addition to SDS-based
delipidation; this protocol was termed ePACT (Treweek et al. 2015). This protocol succeededin expanding slices up to fourfold to visualize Thy1-YFP signals. Despite several distorted cellular architectures, this protocol is a promising approach for scalable visualization ranging from amacroscopic view of tissue slices to a microscopic image with subcellular resolution.

SWITCH-Based Glutaraldehyde
Glutaraldehyde is also commonly used for intermolecular cross-linking of endogenous proteins(Hopwood 1972). The fixing effect of the bifunctional aldehyde is stronger than that of PFA.
However, it is hard to homogeneously fix thick tissues by the fixative due to its low permeability(Hopwood 1967). The Chung group has discovered that glutaraldehyde can penetrate throughlarge organs such as rat and marmoset brains under nonreactive acidic buffer (pH ∼ 3) and canrapidly initiate cross-link reactions inside deeper regions of tissues by replacement in reactive
neutral buffer (pH ∼ 7), in a process termed SWITCH (Murray et al. 2015) (Figure 2). The
SWITCH-based glutaraldehyde fixation is physically and chemically the most stable among all
fixatives compatible with tissue clearing. Interestingly, this fixation also preserves some lipids andeven small molecules such as dopamine after clearing. Glutaraldehyde-fixed tissues are delipidated
by boiling at 80◦ C in aqueous clearing solution containing 200 mM SDS. Nevertheless, due tothe rigid fixation, the intensive treatment does not result in significant loss of proteins or inantigenicity.

PERMEABILIZATION
Permeabilization is an essential step for promoting the substitution of water inside tissues withhigh-RI medium. In this step, cell-permeable molecules are substituted for water driven by osmoticpressure at high concentration (Figure 3). Alternatively, lipids—barriers to molecular flux—areremoved to facilitate passive diffusion of external molecules into tissues (Figure 1). Swellingshrinking behavior of tissues under each chemical treatment may be helpful for understanding
the underlying molecular mechanisms of permeabilization. Most permeabilizing reagents in current protocols could be categorized into one of three groups: (a) water-miscible polar solvents,(b) hyperhydration reagents without delipidation, or (c) delipidation reagents. The delipidation
part includes several strategies for active detergent influx into tissues; one example is electric fieldpotential, which is utilized in CLARITY. According to previous clearing results, fixed tissues could
be fully transparentized by thorough delipidation. Generally, permeabilization by polar solventsor delipidation reagents provides much clearer specimens than that by hyperhydration reagents
without delipidation. Thus, the former reagents are appropriate for comprehensive macroscopic
analysis such as whole-organ or whole-body imaging by LSFM. In contrast, the latter reagents
without delipidation, such as urea and sugar, specialize in the preservation of precise cellular architecture and so are suitable for detailed microscopic observation with subcellular resolution bymultiphoton fluorescent microscopy.

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a PBS

Osmotically balanced

H2O

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c SeeDBb ScaleA2

Tissue expansion

H2O
Urea

Osmotically balanced

H2O
Fructose
Molecular flux is balanced by serial increases
in fructose concentrationd FRUIT, ScaleS

Osmotically balanced

H2O
Urea
Sugar,DMSO

Figure 3
Plausible mechanisms in permeabilization without delipidation by hyperhydration reagents. (a) Brain tissues
in PBS are osmotically balanced. (b) In the ScaleA2 protocol, highly permeable urea may initiate the importof external water molecules by increasing internal osmotic pressure. (c) In the SeeDB protocol, osmotic
dehydration can be adequately balanced with permeation of fructose by controlling gradual increases infructose concentration. (d ) FRUIT and ScaleS achieve rapid clearing of brain tissues without significant
deformation by means of the osmotically balanced potentiation of molecular flux across tissues.

Water-Miscible Polar Solvents
High-RI (n > 1.5) aromatic solvents are immiscible with water, whereas water-miscible polar
solvents such as alcohols and THF are miscible with both aromatic solvents and water in any
proportion (Figure 1). Therefore, solvents can play an essential role in mediating the substitution of water with high-RI aromatic solvents inside tissues. Classically, biological tissues were
dehydrated by alcohols (Spalteholz 1914). In an early protocol from the Dodt group, tissues were
dehydrated by serially increasing the EtOH concentration up to 100% and rinsing with 100%
hexane (Dodt et al. 2007). EtOH is small enough to be rapidly replaced with water by passive
diffusion. Water composes 70–80% of major tissues, forms 3D hydrogen-bonding networks withintracellular constituents, and creates space inside tissues (Pal et al. 2002, Zimmerman & Minton1993). EtOH has much less hydrogen bonding ability than water. Thus, EtOH-solvated tissues
are shrunk and hardened after dehydration. Recently, Ertürk et al. (2012) pointed out that the
EtOH-BABB clearing protocol failed to clear myelinated tissues such as the adult mouse brain.
So EtOH may be insufficient for tissue delipidation. The Dodt group screened more than 10,000
chemicals to find a highly effective and GFP-friendly dehydrating reagent, THF (Becker et al.
2012). This polar ether–based 3DISCO protocol has achieved extremely rapid dehydration due
to its high permeable kinetics and has provided fully cleared organ samples after RI matching.
Because THF has significant lipophilicity (Dı́az et al. 1992), this protocol may accompany highly
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effective delipidation. THF-solvated tissues are more slightly shrunk than EtOH-solvated ones,
probably because THF lacks donating hydrogen. After mounting in DBE, tissues become rapidly
cleared without further shrinkage. The Nemoto group and the Pavone group have investigatedanother water-miscible polar solvent, 2,2 -thiodiethanol (TDE) as a clearing medium (Aoyagi et al.
2015, Costantini et al. 2015). TDE has also been utilized as a water-miscible mounting medium
because of its high RI (n = 1.52), which is equivalent to that of glass and typical immersion oils
(Staudt et al. 2007). So TDE can be expected to combine the chemical properties of both dehydration and RI matching. However, adult mice brains were not fully transparentized and significantly
shrunk after 60% or 97% TDE treatment (Aoyagi et al. 2015). This result may be attributed to
the relatively slow kinetics of TDE influx. The clearing performance by TDE could be potentially
improved by alterations in protocol such as serially increasing TDE concentration or combining
hyperhydration reagents as described below. Overall, permeabilization by polar solvents is a feasible strategy for tissue clearing, but with the drawbacks of handling of harmful chemicals andquenching of fluorescent proteins.

Hyperhydration Reagents without Delipidation
Among hyperhydration reagents, the Miyawaki group discovered the clearing ability of an aqueous
urea–based solution termed Scale (Hama et al. 2011). Although the cell membrane limits molecular
flux, small, uncharged molecules can quickly pass through the lipid bilayer (Petty 1993). A small,uncharged urea molecule (with molecular weight = 60 and molecular radius = 0.22 nm) is oneof the most permeable molecules and has strong hyperhydration abilities due to its bearing bothhydrogen donor and acceptor groups. Also, urea is a relatively weak denaturant for proteins andnucleic acids because it disturbs their hydrogen-bonding networks (Hua et al. 2008, Priyakumar
et al. 2009). Furthermore, urea increases membrane fluidity (Barton et al. 1999, Feng et al. 2002)
to enhance molecular flux across membranes and retain chemicals in cells (Bentley et al. 1997).
The optimized ScaleA2 cocktail contains 4 M urea, 10% glycerol, and 0.1% nonionic detergent
Triton X-100. Once biological tissues are immersed in ScaleA2, tissues begin to swell and optically
clear. One possible hypothesis for tissue expansion is that urea relaxes protein scaffolds involving
solid tissue frameworks such as collagen fibers (Usha & Ramasami 2002), as shown in the dramatic
expansion of collagenase-treated, hydrogel-embedded tissues in ePACT (Treweek et al. 2015).
As a result, solvation of dense fiber proteins could lead to reduced light scattering inside tissues.
In addition, the inside osmotic pressure would be increased, thereby inducing influx of outermolecules, including water and other ingredients (Figure 3). If so, the ionic gradient between
internal and external tissue would also be important in the molecular flux and optical clearing by
urea (Hou et al. 2015). Actually, ionic contamination significantly suppresses both tissue expansion
and clearing by ScaleA2. Furthermore, aqueous guanidine solution, which is similar to urea but
is an ionic chaotrope (Greene & Pace 1974), displayed much less clearing in brain tissues andshrank tissues (T. Murakami & H.R. Ueda, unpublished results). This hypothesis of increased
internal osmotic pressure may also be supported by the empirical observation that tissues swollen
due to ScaleA2 immediately shrank to nearly their original size after PBS washing. We also
note that clearing by urea-based cocktails does not accompany lipid removal inside tissues unless
delipidation chemicals like detergents or aminoalcohols are included. Tissues treated with FRUIT
and ScaleS can be clearly stained with the lipid probe DiI (Hama et al. 2015, Hou et al. 2015).
Taken together, the evidence suggests that urea may initiate the import of external small molecules
into tissues by increases in osmotic pressure inside tissues without disturbing the lipid bilayer
membrane and by relaxation and solvation of highly dense fiber proteins. The Mason group has
developed a formamide-based clearing protocol termed ClearT (Kuwajima et al. 2013). Formamide
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is also a small, uncharged denaturant with both hydrogen donor and acceptor groups. However,
the hydration energy of urea is expected to be much higher than that of formamide becauseof the strong synergetic effect of two NH2 groups, according to a comparison between urea andthe formamide derivative acetamide ( Jedlovszky & Idrissi 2008). Actually, 10-wt% urea solution
exhibited higher swelling ability and clearing performance for the adult mouse brain than did
10-wt% formamide solution (T. Murakami & H.R. Ueda, unpublished result). Nevertheless,
ClearT transparentized whole mice embryos much more rapidly and efficiently than did ScaleA2.
This is mainly because polar formamide considerably dehydrates tissues by serially increasing theconcentration up to 95%. Formamide is an aprotic polar solvent like N,N-dimethylformamide
and DMSO and can solubilize lipids. Therefore, delipidation of tissues by high concentrations offormamide may contribute to the clearing performance of ClearT . Strictly speaking, formamide
should be categorized as a water-miscible polar solvent. But we introduce this denaturant as ahydrogen-promoting solvent to compare it with urea.
Permeabilization by substitution of water with hyperhydration reagents driven by the osmoticpressure difference is a straightforward strategy for preservation of an intact hydration environment inside tissues. In the ancient past, people soaked fruits in syrup for antisepsis to createa translucent glaze. Analogously, the Imai group developed a fructose-based clearing method
termed SeeDB (Ke et al. 2013). In this protocol, fixed tissues are serially incubated in 20%(wt/vol) to 80.2% or 86.7% (wt/wt) fructose solution. SeeDB-treated tissues kept their original sizes throughout the protocol. Osmotic dehydration was adequately balanced with permeationof the small monosaccharide fructose by controlling gradual increases in fructose concentration.
However, the extremely high viscosity of the saturated fructose solution complicates preparation,
handling, and cleanup.
To address this issue, the Jiang group developed less viscous cocktails containing fructose andurea, termed FRUIT (Hou et al. 2015). They also investigated osmotically balanced protocols
without tissue deformation and demonstrated the importance of the gradient in solute concentration and the balance of ionic osmotic pressure. Nevertheless, fructose-based protocols suchas SeeDB and FRUIT cannot avoid Maillard browning discoloration derived from the reactive
ketone group of fructose, even in the presence of reductants such as α-thioglycerol.
The Miyawaki group established a new clearing protocol, ScaleS, by combination of urea andthe monosaccharide sorbitol, which lacks a ketone group (Hama et al. 2015). ScaleS succeeds inthe rapid clearing of brain tissues without significant deformation by means of the osmoticallybalanced potentiation of molecular flux across tissues. Importantly, clearing by ScaleS is free fromtissue discoloration, from quenching of the fluorescent reporter, and from loss of presynaptic
and postsynaptic fabrics. The Miyawaki group also mentioned pretreatment with a cocktail containing 1 mM methyl β-cyclodextrin, 1 mM γ-cyclodextrin, and 1% N-acetyl-L-hydroxyproline;
this cocktail enhanced tissue permeability. Both of these cyclodextrins are used for extraction ofcholesterol from the plasma membrane (Kilsdonk et al. 1995), and N-acetyl-L-hydroxyproline
loosens collagen structures (Hama et al. 2015). Therefore, removal of cholesterol and relaxationof collagen fibers may expedite molecular influx into tissues.

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Delipidation Reagents
As described below, hydrophilic molecules suitable for RI-matching medium such as contrast materials do not tend to diffuse inside intact tissues due to their large molecular size. Thus, tissue delipidation is important for the full permeation of high-RI medium into deep regions of tissue. Amongwater-soluble molecules, detergents solubilize lipids efficiently. Our group attempted to develop
a novel screening system to discover hydrophilic chemical candidates with high lipophilicity
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in biological tissues (Susaki et al. 2014). We used PFA-fixed brain homogenate instead of a wholebrain sample for the screening so that many chemicals could be tested using only one mouse brain.
The homogenate was mixed with 10 wt% of each chemical solution, and turbidity of the resulting mixture at OD600 (optical density measured at a wavelength of 600 nm) was recorded. The
potential solubility of each chemical to brain lipids was expected to correlate with reduction of
the turbidity of the homogenate. We screened 40 chemicals, including polyhydric alcohols, detergents, and hydrophilic small molecules, corresponding to the ingredients of the ScaleA2 solution:
glycerol, Triton X-100, and urea. Interestingly, a series of aminoalcohols are highly effective insolubilizing the homogenate compared with other alcohols. Taking into account clearing performance for hemisphere and quenching of fluorescent proteins, we finally arrived at a cocktail of 25wt% Quadrol, 15-wt% Triton X-100, and 25-wt% urea, termed ScaleCUBIC-1. ScaleCUBIC-1
treatment efficiently extracted phospholipids and cholesterol from organ samples to result in highly
cleared specimens after PBS washing and RI matching. Because the complete mixture had higher
clearing ability than did each individual solution alone and every combinatorial solution of two of
the three components, we concluded that each chemical works independently. Presumably, basic
Quadrol solubilizes phospholipids by electrostatic interaction, whereas Triton X-100 incorporates
lipids into a micelle. Urea may accelerate permeation of other ingredients by increasing internal
osmotic pressure and/or may relax dense fiber proteins as described above. Recently, we developed an improved cocktail, ScaleCUBIC-1A, to further preserve fluorescence of reporters. The
composition of ScaleCUBIC-1A is as follows: 5-wt% Quadrol, 10-wt% Triton X-100, 10-wt%
urea, and 0–25 mM NaCl (Susaki & Ueda 2016; protocol available at http://cubic.riken.jp). In
this new protocol, ionic osmolality by the addition of salt prevents excess sample swelling. We
discovered the novel clearing ability of aminoalcohols, despite a relatively small chemical library.
Further comprehensive screening should expand the number of hydrophilic reagents with highclearing performance and should reveal additional chemical principles for tissue clearing throughdetailed chemical profiling. Our chemical screening concept is also applicable to whole-plantclearing. On the basis of a similar combinatorial method, the Kurihara group developed an opticalclearing cocktail, termed ClearSee (Kurihara et al. 2015), which efficiently elutes chlorophyll asa source of autofluorescence while maintaining fluorescent protein stability. In contrast to the
Scale-based clearing cocktail by the Sherrier group (Warner et al. 2014), ClearSee succeeded in
the considerable removal of chlorophyll inside plant tissue.
All CLARITY-related clearing methods are based on delipidation by the ionic detergent SDS(Figure 1). Detergents very slowly penetrate into tissues, probably because of their large molecular size, as shown in passive CLARITY and PACT. Thus, acceleration of diffusion kinetics ofdetergents inside tissues is required to sufficiently achieve lipid removal in deep tissue. Therefore,
increasing internal osmotic pressure by urea permeation is a feasible strategy, as in ScaleCUBIC-1,
to passively facilitate detergent influx. Alternatively, there are three other strategies for promoting
diffusion of detergents: electric field potential, transcardial perfusion, and thermal energy.
Electric field potential is the most promising approach to reinforce diffusion of ionic detergents.
The Deisseroth group first introduced the electrophoresis-based clearing method CLARITY torender rapid and full clearing of a whole mouse brain (Chung et al. 2013). In this protocol,hydrogel-embedded tissue samples were delipidated by active diffusion of SDS into tissues induced
by an electric field potential. For proper tissue clearing, a homogeneous electric field and constanttemperature and pH in the electric chamber should be maintained (Tomer et al. 2014). Because
for this electrophoretic tissue clearing method there is much room for optimization of conditions
such as electric potential, temperature, electrode, and chamber, many kinds of CLARITY-derived
methods have been reported (Costantini et al. 2015, Epp et al. 2015, Kim et al. 2015, Lee et al. 2014,
Palmer et al. 2015). High-voltage (>50-V) application in static one-directional electric field causes
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significant tissue deformation, although diffusion of charged molecules is theoretically accelerated
in proportion to voltages greater than 10 V. Increased diffusion may occur because applicationof such large electrical forces can also strain endogenous charged biomolecules such as DNA
and proteins. Interestingly, the Chung group revealed that a rotational electric field selectively
electrotransported charged molecules even under high-voltage application and therefore led to
rapid tissue clearing without macroscopic and microscopic deformation (Kim et al. 2015). Althoughdevice design is complicated, this system represents the most rational and efficient electrophoretic
clearing device.
In animals, blood is usually delivered to the body through the circulatory system by the heartpumping. Thus, transcardial perfusion is another strategy to enforce detergent permeation inside tissues. The Gradinaru group first introduced this type of clearing protocol, termed PARS
(Yang et al. 2014). Whole-body clearing by continuous perfusion of SDS-based clearing medium
provided fully transparentized whole-organ samples such as brain, heart, lung, liver, and kidney
after RIMS treatment (Treweek et al. 2015). We have also developed another CUBIC protocol
for the combination of simple immersion and temporal perfusion of half-diluted ScaleCUBIC-1(Tainaka et al. 2014). Both internal clearing by perfusion and external clearing by immersion
allowed for further effective clearing of whole-body samples (Susaki et al. 2015). As a result, the
CUBIC perfusion protocol has enabled whole-body and whole-organ imaging at the single-cellresolution by commercially available LSFM for the first time.
Because solute diffusion kinetics is dependent on temperature, thermal activation of diffusion
appears to be a straightforward strategy (Murray et al. 2015). However, because PFA-fixed tissues
would be damaged by a strong surfactant solution at elevated temperatures, passive clearing protocols are usually conducted below 37◦ C. In other words, the fixation intensity of tissues limits
the thermal diffusion kinetics of detergents. As described above, the Chung group has introduced
SWITCH-based glutaraldehyde fixation. Those highly fixed tissues are successfully resistant toboiling in SDS-based buffer, even at 80◦ C.

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DECOLORIZINGTo achieve whole-organ/whole-body clearing, it is important to decolorize endogenous pigments
such as heme, riboflavin, melanin, and lipofuscin (Tuchin 2015). These pigments usually interferewith observation. Whereas heme and riboflavin are small ligands for endogenous proteins, melanin
and lipofuscin are oxidatively polymerized pigments that are derived from the amino acid tyrosine(Sealy et al. 1980) and lipoproteins (Brizzee et al. 1984), respectively. Generally, those pigments
can be readily bleached by several oxidative treatments (Andersen et al. 2012). In particular, H2 O2 ,one of the most widely used bleaching chemicals, directly degrades the pigment structure (Dowson
1983, Li et al. 2006, Manicam et al. 2014). Among the recent 3D imaging protocols, in iDISCO
such oxidative bleaching was applied to suppress autofluorescence inside tissues (Renier et al.
2014). In spite of versatile availability for pigment bleaching, such harsh oxidation may be fraught
with undesired protein degradation, leading to loss of GFP signals and antigenicity (Alnuami et al.
2008, Steinke & Wolff 2001). Thus, a milder decoloration strategy is required for tissue clearing
to establish multi-imaging systems compatible with small fluorescent probes, fluorescent proteins,and immunological labeling.
To visualize detailed structures in deeper organs, we must overcome light absorbance by heme,
which is one of the most abundant chromophores in the body (Horecker 1943, Weissleder 2001).
However, heme tightly binds to hemoglobin and can be released only in highly acidic (≤pH 2) orhighly basic (≥pH 11) conditions (Kristinsson & Hultin 2004, Teale 1959). Recently, we discovered another unexpected chemical nature of aminoalcohols: tissue decolorization (Tainaka et al.
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20:02014). Q bands (∼500–700 nm) of heme in erythrocytes treated with Quadrol or ScaleCUBIC-1
reagent were considerably changed from those of the hemoglobin-bound form. These results
imply that aminoalcohols may be bound to heme porphyrin instead of oxygen and histidine inhemoglobin; this tendency to bind heme porphyrin may facilitate heme release and may explain the unexpected expansion of the effective pH window for heme release. Additionally, thebuffering capability of Quadrol in moderately basic conditions should also contribute to the decolorizing performance. SDS-based CLARITY protocols also succeeded in decolorizing heme-richorgans (Epp et al. 2015, Lee et al. 2014, Treweek et al. 2015). This is probably because charged
heme molecules could be eluted by electrophoretic force as well as by denaturation of the hemehemoglobin holoenzyme by SDS.
Unlike the case of heme, there has been no feasible method for decolorizing large pigments
such as melanin and lipofuscin, except for harsh bleaching. It is inherently difficult to elute suchpolymerized pigments without degradation of other constituents. Thus, we have to explore alternative strategies to address this issue. In the case of lipofuscin, its strong autofluorescence is theprominent obstacle for optical fluorescence imaging of elderly tissues. Copper(II) sulfate and Sudan Black B efficiently reduced lipofuscin autofluorescence without significant interference withother fluorophores in sectioned tissues (Schnell et al. 1999). The Gradinaru group demonstrated
that Sudan Black B staining is successfully compatible with tissue clearing, fluorescent reporters,
and immunostaining (Treweek et al. 2015). After whole-body clearing by CUBIC, pigments ineyeballs still impede light transmission due to the strong absorbance of light. Thus, decolorizingof melanin remains a challenge.
Several clearing protocols, such as fructose-based SeeDB and FRUIT and glutaraldehydebased SWITCH, cause undesired Maillard-type discoloration of tissues (Hou et al. 2015, Ke
et al. 2013, Murray et al. 2015). The reactive ketone of fructose or aldehyde of glutaraldehyde
initially reacts with the nucleophilic amino group of endogenous constituents to form Schiff bases,
followed by reversible Amadori rearrangement and irreversible polymerization (Leung 1987). The
formation of the heterogeneous polymer melanodin may be facilitated at high temperatures andlow water activity. This discoloration seriously interferes with light transmission. However, suchdiscoloration can be partly suppressed by reduction by α-thioglycerol or sodium sulfite (Ke et al.
2013, Murray et al. 2015), possibly because the reductive amination of Schiff bases would deactivate
the generation of highly reactive aldehyde intermediates to terminate the polymerization.

REFRACTIVE INDEX MATCHING
RI matching is a final step for tissue clearing to homogenize the RI of the entire tissue. Solutes
and solvents bearing an aryl and/or iodo group display higher RI than do other solutes andsolvents. Moreover, the RI of a solution increases depending on the solute concentration. Thus,
high-RI media are generally composed of aromatic solvents or dense aqueous solutions containing
highly water-soluble contrast reagents or polyalcohols like sugars. The optimum RI value for eachbiological tissue depends on the contents and densities of lipids, proteins, and other constituents( Johnsen & Widder 1999, Tuchin 2015). Although current protocols have been optimized formice brains, RIs of the available media range from 1.44 to 1.56, as shown in Table 1. We discuss
RI matching by comparing protocols to give useful chemical insight into the optimum RI value.
Both BABB and DBE are organic RI-matching media with the same RI value (n = 1.56). However, DBE has faster kinetics and higher potency in tissue clearing than does BABB (Becker et al.
2012). These differences may be attributed to a difference in viscosity (DBE viscosity = 5.3 cP;
BABB viscosity = ∼8.8 cP). THF-dehydrated tissues become rapidly transparentized after immersion in DBE. But cleared tissues become opaque again after prolonged incubation in DBE
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without any change in sample size, indicating that the equilibrated RI value (near 1.56) does notexactly match with the RI of THF- and DBE-treated brains. Because it takes a long time to reachequilibrium in efflux of embedded THF while the influx kinetics of DBE balances with the efflux
kinetics of THF, an optimum RI value for brain samples by the THF and DBE clearing is expected
to be lower than 1.56.
Diatrizoic acid [FocusClear, n = 1.46 (Chiang 2002)], Histodenz [RIMS, n = 1.47 (Yang et al.
2014); SeeDB2, n = 1.52 (Ke et al. 2016)], and iodixanol [SWITCH-related RI medium, n =1.47 (Murray et al. 2015)], which are known as contrast reagents, are utilized as hydrophilic RI
media because of their extremely high solubility in water (Figure 1). However, contrast reagent–based RI media require intensive removal of the plasma membrane in the permeabilization step
due to their large molecular size. Because CLARITY-related clearing protocols can achieve full
delipidation of the tissues of interest by various kinds of permeabilization strategies, these RI
media are suitable for such protocols. Among hydrophilic methods, these protocols provide thegreater clearing of specimens. Thus, the optimum RI value of the lipid-removed brain samples
is considered to be approximately 1.46–1.47 [the SeeDB2 cocktail was designed to match therefractive indices of immersion oil (n = 1.518)]. These reagents can also contribute to high-RI
media, even in relatively high water content, in addition to modest viscosity. Therefore, these
media avoid excess tissue shrinkage after equilibrium.
Polyalcohols such as glycerol (n = 1.47), fructose [SeeDB, n = 1.49 (Ke et al. 2013)], sucrose[ScaleCUBIC-2, n = 1.49 (Susaki et al. 2014)], sorbitol [sRIMS, n = 1.44 (Yang et al. 2014);
ScaleS4, n = 1.44 (Hama et al. 2015)], and aprotic DMSO (Economo et al. 2016, Hama et al. 2015)
are also highly soluble in water and are therefore suitable for preparation of high-RI media. Thesereagents have a significant cost advantage over contrast reagents, but sugar-based RI media areinferior in clearing performance. Small glycerol and monosaccharides are suitable for delipidationfree clearing methods such as Scale and SeeDB. Because these protocols cannot result in fullytransparentized brain samples, the optimum RI value of lipid-intact brain tissues is still unclear. In
the CUBIC protocol, the disaccharide sucrose accelerates permeation into lipid-removed tissues by
increasing internal osmotic pressure. Additionally, urea osmotically counterbalances dehydration
by sucrose to escape from both shrinking and swelling in a fashion similar to that of ScaleS.
CUBIC-treated brain samples are clear enough to be applied to LSFM. But the clearing efficiencyof the CUBIC protocol is less than the clearing efficiencies of THF-DBE and CLARITY-related
clearing protocols (Hama et al. 2015, Kim et al. 2015). The lower clearing efficiency of the CUBIC
protocol is probably because lipids in the brain are not fully removed through this protocol. Thismay be why the CUBIC-optimized RI value (n = 1.49) needs to be slightly higher than the RI
value of CLARITY-related protocols (n = 1.46–1.47).

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PRESERVATION OF FLUORESCENT SIGNALS FROMREPORTER PROTEINS
Fluorescent proteins such as GFP from the jellyfish (Shimomura et al. 1962, Tsien 1998) are
well-established and versatile reporter proteins for monitoring gene expression profiles (Chalfie
et al. 1994), protein localization (Nienhaus & Nienhaus 2014), and sensing endogenous molecules(Mehta & Zhang 2011) in a variety of systems. The structure of the GFP chromophore was characterized as p-hydroxybenzylideneimidazolinone (Cody et al. 1993, Shimomura 1979), which isformed by internal cyclization of a Ser-Tyr-Gly tripeptide and 1,2-dehydrogenation of the Tyr(Cubitt et al. 1995). Wild-type GFP displays a pronounced bimodal absorption profile with two
peaks at approximately 400 and 470 nm, corresponding to the protonated and the deprotonatedforms of the chromophore, respectively (Brejc et al. 1997, Dong et al. 2006). Therefore, the730

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protonation state of the chromophore strongly affects fluorescent emission from 488-nm excitation. Most fluorescent proteins, including Venus (Rekas et al. 2002), mFruits (Shu et al. 2006),
mKate (Pletnev et al. 2008), and DsRed (Yarbrough et al. 2001), share similar chromophore structures, and the deprotonated phenolate forms usually exhibit strong fluorescent emission excitedby the desired wavelengths (Ormö et al. 1996). Thus, fluorescence highly depends on pH (Kneen
et al. 1998) and ionic strength (Ward et al. 1982). Also, the organic solvents (Hama et al. 2011),
detergents, and chaotropic denaturants (Stepanenko et al. 2012) used in the clearing protocols
potentially suppress fluorescent signals from GFP derivatives by protein denaturation. We haveto keep in mind the two deactivation mechanisms of GFP derivatives: (a) protonation of thechromophore and (b) denaturation of the protein structure.
Generally, GFP signals are attenuated by dehydration processes such as EtOH-BABB, relatively GFP-friendly THF-DBE, TDE, and ClearT . Even fructose-based SeeDB also somewhat
suppresses GFP fluorescence, possibly due to low water content (Hama et al. 2015). It is inevitable that dehydration progressively denatures GFP and results in the decay of GFP fluorescence. However, suppressed EGFP fluorescence in resin-embedded dehydrated tissues was
dramatically recovered by alkaline buffer solution treatment (Xiong et al. 2014). Also, the Giesegroup demonstrated that EGFP signals were sufficiently preserved through dehydration by pHadjusted 1-PrOH or t-BuOH and remained for more than 100 days in BABB solution at 4◦ C
(Schwarz et al. 2015). For the preservation of GFP signals, it is important to maintain a basic-pH
condition, with pH adjusted to 9.5 with nonionic trimethylamine. Thus, fluorescent suppression
by the protonation of the chromophore is prior to irreversible quenching by protein denaturation, and denaturation can be dramatically reduced, even in organic solvents. Unlike 1-PrOH andt-BuOH, MeOH and EtOH failed to preserve GFP fluorescence by the same pH adjustment.
A small MeOH or EtOH may penetrate the barrel structure to inhibit the deprotonation of thechromophore. These results indicate that GFP signals can be sufficiently preserved through thepreviously reported dehydration protocols by appropriate pH control.
In contrast, GFP fluorescence is considered to be quenched by protein denaturation in
detergent-based protocols such as CLARITY-related methods, PACT-PARS, and CUBIC. The
quenching of GFP is also significantly affected by temperature (dos Santos 2012, Schwarz et al.
2015). Incubation at elevated temperature may denature GFP proteins while promoting tissue
clearing. GFP fluorescence is well preserved between pH 7.0 and 11.5 at room temperature but
is well preserved only at ∼pH 7.5 at 45◦ C (T. Murakami & H.R. Ueda, unpublished result).
ScaleCUBIC-1 is too basic to preserve GFP signals under long incubation times at 37◦ C. The
composition of ScaleCUBIC-1A was modified to avoid quenching of GFP signals without disturbing clearing performance (Susaki & Ueda 2016; for more details, please see http://cubic.riken.jp).
Among the clearing protocols, urea/sorbitol-based ScaleS is the best protocol for ensuring
GFP fluorescence (Hama et al. 2015). Although this cocktail contains high concentrations ofurea, permeated urea is not sufficient to denature GFP proteins.

STAINING OF ENDOGENOUS COMPONENTS
Recent advances in tissue-clearing techniques allowed us to visualize entire organs at single-cellresolution. Early work relating to 3D tissue imaging have mainly used reporter mice expressing
exogenous fluorescent proteins. Reporter mice are particularly suitable for these technologies
because organ samples could be visualized by microscopes just after tissue clearing. However,reporter animals are limited to certain strains and species. Thus, researchers have also intensively
tackled tissue-staining methods by fluorescent dyes and antibodies to label specific and various
molecular markers as listed in Table 2. Unfortunately, these molecules permeate very slowly
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Tissue-staining protocols

Reference

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SampleStaining procedure

Fluorescent dye

Application

Ertürk et al. 2012

3DISCO

ProtocolMouse: brain, spinal
cord, lung,mammary glands,
spleen, lymph node

Diffusion-limited
staining

Cy3 anti-smoothmuscle actin, FITC
lectin, Alexa Fluor
488

Vasculature, tumor
cells, and immune
cells

Renier et al. 2014

iDISCO(3DISCOclearing)

Mouse: brain,kidney, embryonic
head, embryonicspinal cord

Diffusion-limited
staining, antibody
penetration driven
by dehydrationrehydration
process by MeOH
and PBSTO-PRO family,
EdU labeling, CTB

Neuroanatomy;
neuronalpathophysiology;
and neuraldevelopment,
activity, anddegradation

Kuwajima et al.
2013

ClearT ,ClearT2

Mouse embryo

Diffusion-limited
staining

DiI, CTB, Hoechst

Neural tracing

Hama et al. 2011,2015

Scale, ScaleS

Mouse: brain,embryonic brain;human: brain

Diffusion-limited
staining; urea
induces molecularflux

DiI, PP-BTA-1, PI

Vasculature,development,
connectomics, andplaquepathogenesis in
Alzheimer’s disease

Ke et al. 2013

SeeDB

Mouse: brain,embryoDiffusion-limited
staining

DiI

Neural tracing andanatomySusaki et al. 2014,2015; Tainaka
et al. 2014

CUBIC

Mouse: brain, heart,
lung, kidney, liver,spleen, pancreas,
intestine, stomach,muscle, skin, ear,whole body;
marmoset: brain

Diffusion-limited
staining

PI, SYTO 16

Neural activity,
anatomy, andpathology

Chung et al.
2013,
Costantini et al.
2015, Epp et al.
2015, Lee et al.
2014, Tomeret al. 2014

CLARITY

Mouse: brain,
pancreas, liver,kidney, lung,intestine, spleen,muscle, testis;
human: brain

Diffusion-limited
staining

DAPI, PI, SYTO 16

Neural anatomy andpathology, in situ
hybridization,
multiple rounds ofstaining anddestaining

Treweek et al.
2015, Yanget al. 2014

PACT-PARS

Mouse: brain, heart,
lung, kidney, liver,
spinal cord,pancreas, intestine,
tibia, spleen,
adrenal gland,
fallopian tube,
ovary, whole body;
rat: brain, spinal
cord; human:
tumor biopsy,
pancreas

PACT:
diffusion-limited
staining; PARS:
staining bytranscardial
perfusion

DAPI, Hoechst,
SYTO 24, DRAQ5,
acridineorange/methylene
blue,
NeuroTraceTM
530/615 redfluorescent, DyLight
488 lectin, Atto
565–phalloidin,
Alexa Fluor 647
phalloidin

Anatomy, tumor
cells, andpathology; in situ
hybridization(Continued )732

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(Continued )Reference

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ProtocolSampleFluorescent dye

Application

Kim et al. 2015

Stochastic
electrotransport

Mouse: brain, heart,
lung, kidney, liver,intestine

Staining by
stochastic
electrotransport

Staining procedure

TO-PRO-3,
SYTO 16, Dylight
594 tomato lectin

Vasculature andneuroanatomy

Lee et al. 2016

ACTPRESTO(CLARITYbased
clearing)

Mouse: brain, lung,kidney, liver,spleen, intestine,
testis, thymus,
whole body; rat:
brain, embryo;zebrafish: whole
body; rabbit: brain;human: spinal cord

Staining by
centrifugal force(c-PRESTO) or
convection flow(s-PRESTO)DAPI, SYTO 16

Neural tracing, insitu hybridization

Murray et al.
2015

SWITCH

Mouse: brain, heart,
lung, kidney, spinal
cord; rat: brain,
spinal cord;
marmoset: brain;human: brain

Diffusion-limited
staining controlled
by the interaction
between dyes andtheir targets

DAPI, DiD, DyLight
488 lectin

Entire myelinatedfiber tracts inmouse brain, in
situ hybridization,
multiple rounds ofstaining anddestaining

through a tissue matrix entangled with fibrous proteins and other constituents, and it usually takes
a long time to stain thick tissues (over approximately the millimeter scale) uniformly (Kim et al.
2015). To address this issue, we have to gain deeper insight into the principles of permeation
kinetics of external molecules. In this section, we highlight current staining protocols optimized
for individual clearing protocols and discuss key features of permeation kinetics.
Generally, the staining kinetics of fluorescent molecules depends on the diffusion kinetics
and the chemical interactions with endogenous molecules. The diffusion kinetics in the tissue
matrix may be highly affected by the degree of fixation, probably because the degree of fixation
would determine the pore size of polymerized protein networks inside the tissue (Yang et al.
2014). Thus, ensuring sufficient pore size is important so that dyes can spatially pass throughtissues. Because the cell membrane strictly limits molecular flux, permeabilization would also
critically promote the access of dyes to intracellular biomolecules. Dyes that have permeated into
the tissue are surrounded by enormous endogenous molecules. The diffusion of dyes could besubstantially retarded by interactions with such molecules. Therefore, temporal cancellation of
those interactions could dramatically accelerate diffusion into deeper regions. A careful review
of current protocols, including iDISCO, CUBIC, ScaleS, and SWITCH, as well as CLARITYrelated protocols, would shed light on the underlying principles of chemical staining.

iDISCOiDISCO is a rapid staining protocol that uses organic solvents in combination with 3DISCO by
the Tessier-Lavigne group (Renier et al. 2014). A dehydration-rehydration process by MeOH andPBS significantly facilitated antibody penetration into the sample, but the mechanism by which
this occurred was not clear. Perhaps the hydrophobic denaturation process loosened polymerized
protein matrices to expand pore size. In any case, the iDISCO technique allowed for whole-mountlabeling of embryo mice and several adult mice organs and was compatible with 28 antibodies andwww.annualreviews.org • Chemical Principle in Tissue Clearing733

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several fluorescent dyes. Adult mice brains were stained to visualize the projections of dopaminergic neurons and axons of sensory neurons. In addition to neuroanatomy, Renier et al. used
iDISCO for neuronal pathophysiology, including neural development, activity, and degradation.
Unfortunately, 3DISCO-based clearing seems to be unable to decolorize blood-infused tissues,
because significant autofluorescence remains after the process is complete. Renier et al. suggest
peroxide bleaching to overcome this problem, but this treatment may result in loss of antigenicity
and in protein destruction. Currently, longer excitation/emission wavelengths, especially in the
far-red region, are needed for iDISCO images with high signal-to-noise ratio. Because iDISCO
is incompatible with many fluorescent reporters, Renier et al. suggest antibody labeling of eachfluorescent protein. However, passive antibody labeling appears to be too slow to stain whole adultmice bodies homogeneously, so techniques that can utilize fluorescent proteins without antibody
staining are more attractive for WBC profiling.

CUBIC
In the CUBIC protocol, ScaleCUBIC-1 efficiently removes lipids from conventional PFA-fixedtissues. Thus, exogenous molecules easily permeate into cells. CUBIC enabled homogeneous
whole-mount staining of millimeter- to centimeter-sized samples, including major organs of adultmice, primate brains, and whole mice bodies, by several nuclear staining dyes (Susaki et al. 2014,
Tainaka et al. 2014). Nuclear counterstaining is necessary to acquire whole-organ structural images, which can be used for anatomical annotation in computational comparative analyses. Neuralactivities of Arc-dVenus transgenic mice induced by environmental stimulation were also quantified at single-cell resolution. In addition, a comprehensive and statistical analysis of Langerhans
islets (LIs) in the diabetic pancreas revealed that larger LIs are more sensitive to β cell impairment. CUBIC was also applicable to whole-mount immunolabeling of several organs such as theheart, lung, stomach, and intestine under a conventional PBST (2% Triton X-100 in PBS)-based
staining condition. The low concentration of the detergent may contribute to increased permeation kinetics by the disruption of interactions between antibody and endogenous biomoleculesin spite of potential denaturation. However, CUBIC has relatively lengthy protocols in clearing
and staining. The tested antibodies are also limited. These issues will be overcome largely by the
exploration of further effective clearing chemicals and by optimization of permeation kinetics oflabeled molecules.

ScaleS
ScaleS can clear tissue by urea-driven molecular flux tissue without significant disturbance of theplasma membrane (Hama et al. 2015). Thus, urea seems to be necessary for fluorescent molecule
penetration into cells. Hama et al. (2015) established AbScale and ChemScale protocols for antibody and fluorescent dye staining, respectively. Both protocols relied on urea-based staining
solution, but AbScale used a more modest urea concentration to avoid antibody denaturation.
Urea and N-acetyl-L-hydroxyproline also solvate collagen fibers to relax entangled protein networks. These chemical treatments may play a role in the effective permeation of outer molecules
by enlargement of inside pores. Hama et al. also conducted 3D imaging in a mouse model of
Alzheimer’s disease (AD) to establish proof of concept of chemical staining. They observed amyloid β (Aβ) plaques with both the anti-Aβ antibody and the fluorescent probe and evaluatedinflammatory response in an AD mouse brain by double staining of Aβ plaques and microglia.
Although whether AbScale is adaptable to a wide variety of antibodies is unclear, the series ofchemical treatments in Scale appears to be the most likely to preserve intact structures among thecurrently available protocols.
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CLARITY-Related Protocols
The original CLARITY protocol transparentized whole adult brains by electrophoretic delipidation (Chung et al. 2013). Permeabilized brain samples permitted the penetration of various
kinds of antibodies, dye molecules, and even 50-base-pair RNA probes by passive diffusion. Interestingly, immunolabeling of hydrogel-embedded tissues can be recycled by denaturation byionic detergents without fine structural damage or degraded antigenicity. This staining strategy
was also applied to extensively fixed human tissues to visualize and identify neurons and projections. Hydrogel embedding significantly increased preservation of endogenous proteins during
the detergent-based clearing step. But strong fixation also caused high cross-link density in theacrylamide polymer and inhibited the diffusion kinetics of exogenous molecules. The Gradinaru group optimized the contents of the fixative to accelerate passive diffusion of exogenous
molecules (Yang et al. 2014). This group’s continuous perfusion-based PARS protocol mimicks aheart pumping blood and significantly facilitates the permeation of dye molecules into deep tissue
through the vascular system. This protocol also enables staining of antibodies, fluorescent probes,
and RNA probes to various organs, including the brain, heart, lung, liver, and kidney, and has advanced our chemical understanding of tissue clearing and staining (Treweek et al. 2015). However,staining by passive diffusion may be too slow to label large organ samples homogeneously. The
Chung group proposed an alternative rapid staining strategy based on stochastic electrotransport(Kim et al. 2015). A rotational electric field selectively dispersed highly electromobile molecules
without interfering with the endogenous biomolecules. Significantly, all four molecules, eventhough they had different molecular sizes (70 to 2,000 kDa), benefitted from stochastic electrotransport in their electromobility. This strategy achieved homogeneous staining of whole-organ
samples by fluorescent dyes, proteins, and antibodies within 1 day. More recently, the Sun groupreported another staining strategy, termed ACT-PRESTO (active clarity technique–pressurerelated efficient and stable transfer of macromolecules into organs) (Lee et al. 2016). Centrifugal
force (c-PRESTO) or convection flow (s-PRESTO) enabled rapid immunostaining in 100-μmthick sections within 2–3 h, whereas free diffusion requires 1–2 days. Although Lee et al. (2016)
mainly combined their method with CLARITY, the concept could be applicable to other staining
methods.

SWITCHThe principle of column chromatography is helpful for understanding the diffusion kinetics ofdyes into tissue matrices. The mobility of an analyte on a column is determined by the affinityof the analyte to the stationary phase under the eluent. The analyte flows through the column
in an elute buffer but remains on the column in binding buffer. The Chung group established arapid chemical staining concept termed SWITCH (Murray et al. 2015). Dyes first penetrate into
tissues in the SWITCH-OFF solution, which interrupts the interaction between the dyes andtheir target. Then the dyes specifically bind to their target after replacement of the SWITCHOFF solution with a SWITCH-ON solution, which enhances the interaction between the dyes
and their target. This technique enables uniform staining of a lipid probe (DiI) and antibodies,even in glutaraldehyde-fixed brain tissues. Because only 1-mm-thick slices of mice brains were
immunolabeled in the Murray et al. (2015) study, it is unclear whether this protocol will be applicable to larger tissues. Pores in glutaraldehyde-fixed tissue matrices may be relatively small
for antibody penetration. Nevertheless, SWITCH is an important milestone in passive staining because this method revealed a chemical principle for molecular diffusion into the tissue
matrix.
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OUTLOOK

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The growing development of tissue clearing/staining techniques—from the concept of tissue
clearing proposed by Spalteholz 100 years ago to the rapid development of numerous techniques
over the past decade—has increased our understanding of various biological events in different
organs. In the future, technical challenges might focus on clearing entire rodent bodies or primate
organs. The understanding of the chemical principles of tissue clearing/staining will accelerate
WBC profiling, which will enable identification of cellular circuits across multiple organs and allow
for analysis of cellular dynamics in stochastic and proliferative cellular processes. The journey into
the inner space of mammalian body may result in amazing insights into the fundamental questionof what is health.

FUTURE ISSUES
1. Alternative fixation methods capable of preserving proteins without dense packing oftissue matrix will offer the considerable benefits of compatibility of high clearing performance, prevention of quenching of fluorescent proteins, and permeability of staining
molecules.
2. Comprehensive chemical profiling for tissue clearing will elucidate the chemical properties necessary for delipidation, decolorizing, RI matching, and other potential factors.
3. Although gel-based feeding of mice improves gastrointestinal interference with lighttransmission, these methods are inappropriate for long-term breeding. Nutritious feeds
compatible with tissue clearing will facilitate whole-abdominal imaging.
4. PACT-deCAL and EtOH-BABB were used to successfully clear dissected bone tissues.
More refined protocols that enable both delipidation and decalcification will enablevisualization of the whole cardiovascular system and entire neural networks.
5. The skin could be transparentized by several clearing protocols. But skin substantially
blocks permeation of media and disrupts clearing of inner tissues. Skin-permeable clearing reagents are needed to achieve whole-body imaging of the entire animal body.
6. Mild decolorizing chemistry will be required for highly polymerized melanin pigmentsin the skin, eyeball, hair, and some tumor cells without destruction of other endogenous
constituents.
7. For high-throughput or large screening, it will be necessary to develop cost-effective
labeling technologies that enable homogeneous whole-body staining with fluorescentdyes, fluorescently labeled antibodies, and nucleic acid probes.

DISCLOSURE STATEMENTRIKEN has filed a provisional patent application based on the CUBIC works.

ACKNOWLEDGMENTS
We thank the lab members at the University of Tokyo, E.A. Susaki, and A. Millius for their kind
help in preparing this article. This work was supported by the Program for the Brain Mappingby Integrated Neurotechnologies for Disease Studies from the Ministry of Education, Culture,
Sports, Science and Technology (MEXT) of Japan; by the Program for the Basic Science and736

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Platform Technology Program for Innovative Biological Medicine from MEXT; by the Program
for the AMED ( Japan Agency for Medical Research and Development)-CREST (Core Research
for Evolutional Science and Technology) from MEXT; by a Grant-in-Aid for Scientific Research(grant 25221004) from Japan Society for the Promotion of Science ( JSPS); by a Grant-in-Aid for
Scientific Research on Innovative Areas (grant 23115006) from JSPS; by strategic programs forR&D (President’s discretionary fund) of RIKEN; by an intramural Grant-in-Aid from RIKEN
Quantitative Biology Center; and by the Tokyo Society of Medical Science.

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Contents

Annual Review
of Cell andDevelopmental
Biology
Volume 32, 2016

The Heidelberg Screen for Pattern Mutants of Drosophila: A Personal
Account
Eric Wieschaus and Christiane Nüsslein-Volhard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
Tissue and Organ Initiation in the Plant Embryo: A First Time forEverything
Joakim Palovaara, Thijs de Zeeuw, and Dolf Weijers p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p47
Transcriptional Control of Developmental Cell Behaviors
Yelena Bernadskaya and Lionel Christiaen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p77
Genotypes, Networks, Phenotypes: Moving Toward Plant Systems Genetics
Takehiko Ogura and Wolfgang Busch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 103
Neurogenesis and Gliogenesis in Nervous System Plasticity and Repair
Jonas Frisén p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 127
Phosphoinositides in Control of Membrane Dynamics
Kay O. Schink, Kia-Wee Tan, and Harald Stenmark p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 143
Cytoplasmic Streaming in the Drosophila Oocyte
Margot E. Quinlan p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 173
Cargo Capture and Bulk Flow in the Early Secretory Pathway
Charles Barlowe and Ari Helenius p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 197
The Lysosome as a Regulatory Hub
Rushika M. Perera and Roberto Zoncu p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 223
TFEB and TFE3: Linking Lysosomes to Cellular Adaptation to Stress
Nina Raben and Rosa Puertollano p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 255
Endoplasmic Reticulum–Plasma Membrane Associations: Structuresand Functions
Alessandra Gallo, Christian Vannier, and Thierry Galli p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 279
Signaling and Polarized Communication Across the T Cell
Immunological Synapse
Michael L. Dustin and Kaushik Choudhuri p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 303
vii

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TCR Signal Strength and T Cell Development
Nicholas R.J. Gascoigne, Vasily Rybakin, Oreste Acuto, and Joanna Brzostek p p p p p p p p p p 327
The Cytoophidium and Its Kind: Filamentation andCompartmentation of Metabolic Enzymes
Ji-Long Liu p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 349
How Bacteria Subvert Animal Cell Structure and Function
Alyssa Jimenez, Didi Chen, and Neal M. Alto p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 373

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Metabolism and the Control of Cell Fate Decisions and Stem Cell
RenewalKyoko Ito and Keisuke Ito p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 399
Cell Competition: Mechanisms and Physiological Roles
Cristina Claverı́a and Miguel Torres p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 411
Functions and Regulation of Programmed Cell Death in Plant
Development
Anna Daneva, Zhen Gao, Matthias Van Durme, and Moritz K. Nowack p p p p p p p p p p p p p 441
Focal Adhesion–Independent Cell Migration
Ewa K. Paluch, Irene M. Aspalter, and Michael Sixt p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 469
Plasticity of Cell Migration In Vivo and In Silico
Veronika te Boekhorst, Luigi Preziosi, and Peter Friedl p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 491
Mechanical Control of Epithelial-to-Mesenchymal Transitions in
Development and Cancer
Laralynne Przybyla, Jonathon M. Muncie, and Valerie M. Weaver p p p p p p p p p p p p p p p p p p p p 527
Cellular and Molecular Mechanisms of MT1-MMP-Dependent
Cancer Cell Invasion
Antonio Castro-Castro, Valentina Marchesin, Pedro Monteiro,
Catalina Lodillinsky, Carine Rossé, and Philippe Chavrier p p p p p p p p p p p p p p p p p p p p p p p p p p p 555
Structural Perspectives on Axon Guidance
Elena Seiradake, E. Yvonne Jones, and Rüdiger Klein p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 577
The Role of Adipocytes in Tissue Regeneration and Stem Cell Niches
Brett Shook, Guillermo Rivera Gonzalez, Sarah Ebmeier, Gabriella Grisotti,
Rachel Zwick, and Valerie Horsley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 609
Hemodynamic Control of Endothelial Cell Fates in Development
Guillermo Garcı́a-Cardeña and Bendix R. Slegtenhorst p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 633
Regulation of Hematopoiesis and Osteogenesis by Blood
Vessel–Derived Signals
Saravana K. Ramasamy, Anjali P. Kusumbe, Tomer Itkin, Shiri Gur-Cohen,
Tsvee Lapidot, and Ralf H. Adams p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 649

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Contents

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ARI

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7:30

Lymphangiogenesis: Origin, Specification, and Cell Fate
Determination
Noelia Escobedo and Guillermo Oliver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 677
Emergence and Evolution of Secondary Lymphoid Organs
Harold R. Neely and Martin F. Flajnik p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 693

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Chemical Principles in Tissue Clearing and Staining Protocols for
Whole-Body Cell Profiling
Kazuki Tainaka, Akihiro Kuno, Shimpei I. Kubota, Tatzya Murakami,
and Hiroki R. Ueda p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 713
IndexesCumulative Index of Contributing Authors, Volumes 28–32 p p p p p p p p p p p p p p p p p p p p p p p p p p p 743
Cumulative Index of Article Titles, Volumes 28–32 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 747
ErrataAn online log of corrections to Annual Review of Cell and Developmental Biology articles
may be found at http://www.annualreviews.org/errata/cellbio

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