Hama NatNeurosci2011 Scale

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nature neuroscience advance online publi cation 1

t e c h n i c a l r e p o r t s

1Brain Science Institute, RIKEN, Wako-city, Saitama, Japan. 2School of Life Science, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo, Japan. 3Life

Function and Dynamics, Exploratory Research for Advanced Technology, Japan Science and Technology Agency, Wako-city, Saitama, Japan. 4Tokyo Institute of

Technology, Meguro-ku, Tokyo, Japan. Correspondence should be addressed to A.M. ([email protected]).

Received 21 March; accepted 12 August; published online 30 August 2011; doi:10.1038/nn.2928

imaging is prevented by tissue opacity and light scattering. At present,

standard laser-scanning confocal microscopy (LSCM) penetrates only toa depth of ~150 mm below the brain surface. Two-photon excitation fluo-

rescence microscopy (TPEFM) offers improved depth, but it cannot, even

under ideal conditions, penetrate more than 500800mm from the brainsurface59. Thus, optical sectioning of intact tissue is believed to be insuf-ficient to image and reconstruct large brain projections and cell popula-

tions that are often several millimeters in scale and deep below the surface.Light scattering can be reduced by optical clearing, which aims to

increase tissue transparency to achieve refractive uniformity through-out the specimen and allow greater depth of imaging. Although this

approach is only applied to fixed specimens, it can in principle facilitateoptical sectioning and enable three-dimensional imaging and recon-

struction. Several clearing solutions have been described. The water-soluble reagent FocusClear has been used to treat insect brains1012.

Unfortunately, this commercial reagent is prohibitively expensive forlarger samples. Furthermore, because its contents are proprietary,

FocusClear cannot be optimized for different biological samples. BABB(a mixture of benzyl-alcohol and benzyl-benzoate) is another clearing

solution that has been used with ultramicroscopy13,14 and TPEFM15 toperform three-dimensional reconstructions in whole organs of mice

and fruit flies. In this method, a fixed sample is incubated in BABB afterdehydration with ethanol and hexane. However, the extent to which

this organic chemical quenches fluorescent proteins inside specimensremains unknown. In addition, a technique was reported for clearing

thick slabs of mouse cortex for TPEFM with an index-matched solu-

tion of 60% sucrose16. However, this sucrose-based method confers onlymodest transparency on tissue samples.

We developed a clearing reagent called Scale that alleviates the majorlimitations of previously reported solutions. We found that Scale ren-

ders mouse brain and embryos transparent while completely preserv-

ing fluorescent signals from labeled cells. This combination allows theimaging of intact brain at a depth of several millimeters and large-scalereconstructions of neuronal populations and projections at subcellular

resolution. We demonstrated proof of principle by reconstructing net-works involving cortical, callosal, hippocampal and neurogenic popula-

tions. We also developed additional Scale reagents and protocol variantsfor specific experimental applications, and discuss potential future

applications for quantitative three-dimensional brain reconstructions.

Optical methods for viewing neuronal populations and

projections in the intact mammalian brain are needed, but

light scattering prevents imaging deep into brain structures.

We imaged fixed brain tissue using Scale, an aqueous reagent

that renders biological samples optically transparent butcompletely preserves fluorescent signals in the clarified

structures. In Scale-treated mouse brain, neurons labeled with

genetically encoded fluorescent proteins were visualized at

an unprecedented depth in millimeter-scale networks and at

subcellular resolution. The improved depth and scale of imaging

permitted comprehensive three-dimensional reconstructions

of cortical, callosal and hippocampal projections whose extent

was limited only by the working distance of the objective lenses.

In the intact neurogenic niche of the dentate gyrus, Sca le

allowed the quantitation of distances of neural stem cells to

blood vessels. Our findings suggest that the Scale method will

be useful for light microscopybased connectomics of cellular

networks in brain and other tissues.

An important challenge in biological research is the development of high-resolution optical methods to label and image cell populations in three

dimensions deep in intact tissue. For example, the ability to image andreconstruct intact neuronal networks would be valuable for understand-

ing structure-function relationships in the brain. Methods for labelingspecific cell types regardless of tissue depth and geometry have rapidly

progressed with the advent of genetically encoded fluorescent proteinsand transgenic marking methods such as Brainbow1. However, com-plementary optical techniques to image and reconstruct labeled three-

dimensional cell populations deep in intact tissue are also needed.Three-dimensional imaging of biological tissue typically involves

mechanical sectioning to improve axial resolution and access to deeper

structures. However, although this approach can have a high degree ofoptical resolution, promising current methods, such as array tomogra-phy and serial block-face scanning electron microscopy, are costly and

laborious, require sophisticated data reconstruction procedures and arecurrently limited to smaller tissue volumes24. In contrast, optical sec-

tioning provides a potentially fast, simple and inexpensive alternative forthree-dimensional reconstruction of fluorescently labeled structures at

subcellular resolution. However, the utility of optical sectioning for deep

Scale: a chemical approach for fluorescence imagingand reconstruction of transparent mouse brain

Hiroshi Hama1, Hiroshi Kurokawa1,2, Hiroyuki Kawano1,3, Ryoko Ando1, Tomomi Shimogori1,Hisayori Noda1,4, Kiyoko Fukami2, Asako Sakaue-Sawano1,3 & Atsushi Miyawaki1,3
mailto:[email protected]:[email protected]://www.nature.com/neuro


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2 advance online publ ication nature neuroscience


phosphate-buffered saline (PBS), 60% sucrose/PBS, FocusClear,

MountClear and ScaleA2. Transmission was measured in a spectro-photometer with water as a reference sample. The ScaleA2-treated slice was

substantially more permissive to visible and infrared light (350920 nm)than slices incubated in other reagents (Fig. 1b).

We next incubated intact fixed mouse brain in a Sca leA2 solution.Incubation for >2 weeks substantially cleared brain tissue; transparency

was evident against a patterned background (Fig. 1c) or by penetration

with a 532-nm laser light (Fig. 1d). The tissue-clearing effect of ScaleA2was also prominent on whole mouse embryos (Fig. 1e).

To estimate the extent of brain expansion, we calculated brain volume

by liquid displacement before and after ScaleA2 treatment by slowlylowering samples into a graduated cylinder containing water. The mean

sample volume was doubled (197 13%, n = 5) by ScaleA2 treatment.Taking the cube root of 1.97, we assume that ScaleA2 causes a 1.25-fold

linear expansion. To characterize the expansion of macroscopic struc-tures in two dimensions, we time-lapse imaged fixed brain slices (1 mm

thick) of a transgenic mouse line, thy1-YFPline H (YFP-H)17,18 duringa 5-d incubation in ScaleA2 solution (Fig. 1fyand SupplementaryFig. 2). We traced the outlines of the slices and several internal struc-tures in transmitted-light bright-field and fluorescence images. Overlay

RESULTS

Development and properties of the Scale reagent

The discovery of the Scale reagent was based on a serendipitous observa-

tion. We found that polyvinylidene fluoride membranes became transpar-ent when soaked in 4 M urea, which promotes the hydration of biological

samples (Supplementary Fig. 1). This result inspired us to search for anoptimal reagent to clear fixed biological samples for light microscopy.

We first treated mouse brain sections (60 mm thick) fixed with 4% para-

formaldehyde (PFA) with solutions containing 18 M urea. After 48 h,sections treated with 48 M urea became transparent along with someexpansion. To further optimize tissue clearance, we next combined urea-

containing solutions with other ingredients. The most effective solution,which we named ScaleA2, was composed of 4 M urea, 10% (wt/vol) glyc-

erol and 0.1% (wt/vol) Triton X-100. Glycerol was predicted to preventexcess hydration and minimize tissue expansion. ScaleA2 has a pH of 7.7

and refractive indices of 1.382, 1.387 and 1.380 at 589, 486 and 656 nm,respectively. ScaleA2 is colorless; the solution absorbs light at 276 nm but

is permissive to light greater than 300 nm (Fig. 1a).To examine tissue transparency quantitatively, we measured transmis-

sion in brain slices. We prepared 1.5-mm-thick slices from mouse brainsamples that had been fixed and incubated in water-soluble reagents:

Figure 1 Tissue clearing performance of ScaleA2. (a) Transmission curves of ScaleA2 (blue), 60% sucrose/PBS (green), FocusClear (yellow) and MountClear

(magenta). (b) Transmission curves of fixed brain slices (1.5 mm thick) in ScaleA2 (blue), 60% sucrose/PBS (green), Focus/MountClear (magenta, a slice treated

with FocusClear was placed in MountClear) and PBS (violet) after treatment with the respective solutions. (c,d) A whole fixed and cleared brain of a mouse

(P15) after treatment with ScaleA2 for 2 weeks. (c) A photo was taken with a black and white pattern as background. (d) The green light from a 1-mW, 532-nm

laser beam pointer traversed the cleared brain. (e) A photo of two embryos (E13.5) taken with a black and white pattern as background. Left, embryo placed in

PBS after fixation with 4% PFA. Right, embryo incubated in ScaleA2 solution for 2 weeks after fixation with 4% PFA. (fy) Characterization of the expansion of

macroscopic structures in fixed brain slices of a YFP-H mouse during ScaleA2 treatment. A coronal slice (1 mm thick) containing the hippocampus was prepared

from a 9-week-old mouse. The slice was split into two halves and the right half was incubated in ScaleA2 solution for 5 d while the left half was incubated in

PBS. Before (0 d, fi) and 1 d (jm), 2 d (nq) or 5 d (ru) after these incubations, the pair of slices on a coverslip with a patterned background were imaged

using a fluorescence stereomicroscope for transmission (f, i, j, m, n, q, r and u) and YFP fluorescence (g, h, k, l, o, p, s and t). The slice became transparent

and expanded after a 12-d incubation in ScaleA2 solution (l, m, p, q, t and u). The extent of the linear expansion was calculated as 1.28. Ag, amygdala; Cp,

cerebral peduncle (basal part); Cx, cortex; Dmn, dorsomedial nucleus; Hf, hippocampal formation; Pmc, posteromedial cortical amygdala nucleus. The outlines

of the slices and their internal structures at 0 d and 5 d were drawn with blue and orange, respectively. The outlines of the PBS-treated slice at 0 d and 5 d

overlapped substantially (v). Reduced drawings of the outlines of the ScaleA2-treated slice at 5 d also overlapped with the outlines at 0 d extensively (w). In

addition, the outlines of the ScaleA2-treated half (green) at 0 d were inverted and overlaid to the outlines of the PBS-treated half (magenta) at 0 d. As the brain

slice had been split slightly asymmetrically, the edges of each half were not precisely even, but proper alignment was achieved (x). A similar overlay was done

between the size-normalized outlines at 5 d (y). In x and y, the difference between green and red traces indicates the inherent baseline left/right asymmetry of

the slice. Notably, the degree and distribution of the asymmetry are almost identical between x and y. All scale bars represent 5 mm.

PBS

a

Wavelength (nm)

100

80

60

40

20

0Transmittance(%)

ScaleA2

ScaleA2

ScaleA2PBS

b

Before

1

d

2

d

5

d

Transmission TransmissionYFP YFP

Before5 d

Hf

Ag

Cp

Cx

Dmn

Pmc

f g h i

r s t u

n o p q

j k l m

v w yx

PBSScaleA2(inverted)

60

40

20

0Transmittance(%)

800700600500400300

Wavelength (nm)

60% sucrose/PBSFocusClearMountClear

ScaleA260% sucrose/PBSFocus/MountClearPBS

c d

e900800700600500400300

900


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and several GFP-like proteins was resistant to 4 M urea at pH 7.7(Supplementary Fig. 4a,b).

To examine the in vivo stability of EGFP fluorescence in ScaleA2, wetransfected the protein into cultured HeLa cells. After fixation, fluores-

cent cells were time-lapse imaged in a ScaleA2 solution and no substantialdecrease in fluorescence intensity was observed (Fig. 2a). In contrast,

when cells were incubated in BABB following dehydration with ethanol

and hexane13,14, EGFP fluorescence diminished over time (Fig. 2b).Next, we applied ScaleA2 to fixed brains of the YFP-H line17,18, in

which yellow fluorescent protein (YFP) is expressed in a subpopulation

of neurons confined to pyramidal neurons in the hippocampus andneurons with somata in layers V and VI in the cerebral cortex. The

whole fixed brain showed homogeneous fluorescence under blue light.

comparisons between Scaled and control samples revealed that the slice

outlines and relative positions of internal structures (hippocampus,amygdala and white matter) maintained their overall shape and pro-

portions, indicating that tissue expansion was isotropic and homog-

enous. Similar experiments were performed using 50-mm-thick brainslices of another transgenic mouse line, thy1-GFPline M (GFP-M)17

(Supplementary Fig. 3). Although ScaleA2-treated samples were typi-

cally soft and fragile, macroscopic structures that maintained their pro-portions in slice samples lacking full connective tension should showeven better preservation of topology in intact, unsectioned whole brains.

Fluorescence imaging with the Scale reagent

A critical question for brain clearing solutions is whether the capabil-ity for fluorescence imaging is retained. It was reported that wild-type

Aequorea green fluorescent protein (GFP)is sensitive to 8 M urea at acidic pH but not

at neutral or alkaline pH19. We verified thatthe fluorescence of enhanced GFP (EGFP)

60

ScaleA2

Time (min)50403020100 6050403020100

1.0

0.5

0.0

1.0

0.5

0.0

Normalized

fluorescenceintensity

Time (min)

50% ethanol

80% ethanol

100% ethanol

Hexane

BABB

thy1-YFP line H

BABB

a b c

ScaleA2

Figure 2 Comparison of ScaleA2 with BABB.

(a,b) Sensitivity of EGFP fluorescence to ScaleA2

solution and a conventional chemical clearing

reagent (BABB). Cultured HeLa cells expressing

EGFP were fixed with 4% PFA and were time-

lapse imaged while being exposed to ScaleA2

solution (a) or BABB following dehydration with

ethanol and hexane (b). Replacement of Hanks

Balanced Salt Solution with ScaleA2 resulted

in a change in focus and a slight decrease influorescence intensity. (c) Fluorescence images

comparing the preservation of YFP signals

between aqueous (left) and chemical (right) clearing agents. The brain of a YFP-H mouse (7 weeks old) was split into two halves. The left half was treated

with ScaleA2 for 3 d. The right half was treated with BABB after dehydration. Then slices (1 mm thick) were prepared and imaged for fluorescence with a

stereomicroscope. The original shape of the fixed brain is drawn with broken lines. Scale bar represents 5 mm.

2P 1P

2.0mm

(1.6mm)

d

e

f

i

j

k

g h

Pial surface

V

CA1White matter

VI

2.0 mm(1.6 mm)

2.0 mm

xy

z

920-nm laser

thy1-YFP line H

(3 weeks old)

a b c

Hippocampus

Cerebralcortex

Pial surface

VVI

CA1

DG

White matter

IV

thy1-YFP line H

(13 weeks old)

line

Objective

lens

xy

z

920-nm laser

4.0mm

l m

3.3mm

DG

CA3

CA1

White matter

Hilus

GCLML

on

4.0 mm(3.2 mm)

Excised hippocampusof thy1-YFP line H

(13 weeks old)

Objective

lens

920-nm laser

z

xy

Caudal

Anterior

Posterior

Dorsal

Rostral

(2.6mm)

Objective

lens

Cerebralcortex

Hippocampus

Figure 3 Three-dimensional reconstructions ofYFP-expressing neurons in ScaleA2-treated brain

samples of YFP-H mice. The actual imaging

depth is shown in parentheses. Unsectioned

brains (am) and an excised hippocampus (n,o)

were imaged. (ac) TPEFM imaging using a

25 objective (XLPLN25XWMP, numerical

aperture (NA) = 1.05, working distance =

2.0 mm). The experimental setup for TPEFM

imaging using the commercially available

objective is shown in a. A three-dimensional

reconstruction of YFP-expressing neurons in 16

(8 2) quadratic prisms located in the cerebral

cortex and hippocampus is shown in b.

A high-magnification xyimage at a depth of

0.9 mm (a yellow box in b) is shown in c.

(dk) Three-dimensional reconstruction ofYFP-expressing neurons in a quadratic prism

located in the cerebral cortex. The same brain

region was imaged using a 20 objective

(W-PlanApochromat, NA = 1.0, working distance

= 2.0 mm) and taking both two-photon (920-nm excitation, dg) and one-

photon (514-nm excitation, hk) approaches. For each volume rendering,

three xyimages at different zpositions (df and ik) are presented.

(lo) TPEFM imaging using a custom-designed objective with a working

distance of 4.0 mm. The experimental setup for TPEFM imaging using the

objective lens is shown in l and n. Three-dimensional reconstructions of

YFP-expressing neurons in a quadratic prism located in the cerebral cortex

and hippocampus (m) and in 24 (4 6) quadratic prisms located in the

excised hippocampus (o) are shown. DG, dentate gyrus; GCL, granule cell

layer; ML, molecular layer. All scale bars represent 50 mm.


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(Fig. 3df). The imaging depth was sufficient

to reach the dorsal tip of the hippocampus(Fig. 3g). We then imaged the same region with

a 514-nm excitation and an internal descannedpath detector through a confocal pinhole (~2

Airy disks; Supplementary Fig. 6b). This one-photon excitation imaging setup produced

sufficiently bright images at depths permittedby the objectives working distance (~2.0 mm;

Fig. 3hk), suggesting that very little scatteringoccurred inside the specimen. In deep regions,

however, TPEFM yielded a better signal-to-noise ratio than LSCM.

A similar three-dimensional reconstruc-tion was performed by TPEFM using an older

YFP-H mouse (13 weeks old; Supplementary

Video 1). Axons that traveled horizontally

through the dendritic trees of the cortex wereidentified and individual axons tunneling

inside white matter was also discernable.

Beyond the current imaging depth limitWith brains cleared by ScaleA2, the imaging depth limit was deter-

mined by the working distances of currently available objective lenses.Among commercially available lenses, for example, a 40 objective

(LUMPLFLN40XW, NA = 0.80, working distance = 3.3 mm) has alonger working distance but a lower NA. Although the use of this lens

permitted an imaging depth of 3 mm (Supplementary Fig. 7a,b), theresolution was lower. We therefore asked the manufacturer (Olympus)

to develop a customized 25 objective lens, which has a longer work-ing distance (4 mm) and a sufficiently high NA (1.0). Using this lens,

we were able to generate very long quadratic prisms of the YFP-H linebrain, with reconstructions that extended from the brain surface to the

dentate gyrus (Fig. 3l,m and Supplementary Video 2).

In addition to reaching new depth limits for brain reconstruction offluorescent neurons, we were also able to optically reconstruct exten-sive neuronal networks. The hippocampal formation was excised from

a fixed and cleared YFP-H brain to permit a comprehensive three-dimensional reconstruction of the hippocampus containing the den-

tate gyrus and Ammons horn fields (Fig. 3n,o). Fine structures in thesame excised hippocampal preparation were visualized by increasing

the photomultiplier tube sensitivity (Supplementary Fig. 7ce).

Surveying commissural axons in the intact mouse brain

To determine whether the Scale system allows a comprehensive per-

spective of specific axon projections, we used a macro-confocal micros-copy system (AZ-C1, Nikon) and imaged optically cleared brains in

which specific neurons had been fluorescently labeled. We focused on

axons in the corpus callosum, which connects the left and right cere-bral hemispheres21,22. Tracing their axon bundles across the midlinerequires laborious procedures that produce a large number of sections.

To visualize callosal axon projections of layer II/III pyramidal neu-rons, we electroporated plasmids encoding EYFP in utero into the

dorsal ventricular zone of the mouse forebrain at embryonic day 15.5(E15.5)22,23. The embryos were raised until postnatal day 10 (P10), when

callosal connnections are almost fully established21,22, and their brainswere removed and fixed. After incubation in ScaleA2 for 1 week, the

entire EYFP signal in the brain sample was collected using a 1 objective(AZ-PlanApo, NA = 0.1, working distance = 35 mm). The zstack image

(Fig. 4a) mapped the labeled pyramidal neurons in layers II and III in theipsilateral (right) hemisphere and provided sweeping views of fluorescent

We then cut it at midplane and incubated the left half in Sca leA2 for3 d while dehydrating the right half with ethanol and treating it withBABB13,14. Yellow fluorescence was preserved in the ScaleA2-treated

half but not in the half exposed to ethanol and BABB (Fig. 2c). In addi-tion, the ScaleA2-treated half expanded, whereas the BABB-treated half

shrunk. A similar comparison with consistent results was made usinga fixed brain sample from the GFP-M line17 (Supplementary Fig. 5).

Three-dimensional reconstruction of neuronal structures

We examined the three-dimensional architecture of neuronal networkscomprised of fluorescent neurons from the fixed and cleared intact brain

of a YFP-H mouse using TPEFM with 920-nm excitation (Fig. 3a). Witha TPEFM system (Olympus FV1000MPE) employing a 25 objective

(XLPLN25XWMP, numerical aperture (NA) = 1.05, working distance =2.0 mm) and correction collar, an imaging depth of 2 mm was achieved

(Fig. 3b). The three-dimensional reconstruction extended from the cere-bral cortex to the dorsal tip of the CA1 region through the white mat-

ter (corpus callosum). Cortical layer V/VI pyramidal neurons and theirdendritic networks were well resolved; individual dendritic spines were

discernable in an expanded view at a depth of 0.9 mm (Fig. 3c). Given theestimated 1.25-fold expansion along one axis, the imaging depth in real

tissue can be obtained by multiplying the measured depth value by 0.8.In these experiments, imaging at an xy position produced a data

unit in the shape of a long quadratic prism and three-dimensionalreconstructions were extended in the xy plane. To reconstruct corti-

cal networks, multiple units of data were generated at neighboringxy positions and combined using the microscope systems tiling

software (Fig. 3b).

One- and two-photon microscopy of Scaled brain

One-photon excitation fluorescence microscopy should benefit from

tissue clearing to an even greater extent than TPEFM. To illustrate this,we employed LSCM to image a ScaleA2-treated brain sample. We exam-

ined the three-dimensional structures of a 3-week-old YFP-H mouseusing a ZEISS LSM710-NLO system equipped with a 20 objective

(W-PlanApochromat, NA = 1.0, working distance = 2.0 mm). With 920-nmexcitation and an external non-descanned detector (Supplementary

Fig. 6a), which is common to TPEFM20, three-dimensional reconstruc-

tion of the cortex was achieved to a depth of 2.0 mm. The high resolutionof the reconstruction was demonstrated byxy images at various depths

R L

CC

CxII / III ML

LVLV

Cx

CPuCPu

aCx

HC

TM

LbCx

HC

Lc

Figure 4 Visualization of labeled callosal connections in the intact mouse brain. A population of

layer II/III pyramidal neurons was labeled by in uteroelectroporation of plasmids encoding EYFP into

the dorsal ventricular zone on the right side (R) of the mouse forebrain at E15.5, and their axonal

projections into the left side (L) were visualized at P10 using a macro-zoom confocal microscope

after fixation and a 7-d treatment with ScaleA2. CC, corpus callosum; CPu, caudate putamen; Cx,

cortex; HC, hippocampus; LV, lateral ventricle; ML, midline; TM, thalamus. (a) We acquired 18

confocal images (52-mm steps) using a 1 objective lens at scanner zoom 3, and zstacked them.

(b) We acquired 17 confocal images (43-mm steps) using a 2 objective lens at scanner zoom 2,

and zstacked them. (c) We acquired 34 confocal images (10.8 mm steps) using a 2 objective lens

at scanner zoom 4, and zstacked them. All scale bars represent 500 mm.


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Scale reagent variants for targeted applications

ScaleA2 is an optimal all-purpose solution for most brain clearing

experiments. Nevertheless, we optimized other Sca le variants that

could provide better control of certain properties of cleared tissue forspecific applications.

An ideal clearing agent will preserve the volume of tissue for more

accurate reconstruction. However, BABB and FocusClear make biologi-cal samples shrink, while ScaleA2 causes expansion. Thus, we devel-

oped a modified solution, ScaleU2, composed of 4 M urea, 30% glyceroland 0.1% Triton X-100. ScaleU2 requires longer incubation times to

achieve clearing, but it reduces tissue expansion and the fragility ofcleared brain samples and is therefore suitable for soft samples such

as mouse embryos. To assess the utility of ScaleU2, we analyzed cell-cycle profiles during mouse embryogenesis in a transgenic mouse line

(#596/#504) that expresses the Fucci probe in which almost every cellnucleus exhibits either red (G1/G0) or green (S/G2/M) fluorescence

27.

We incubated fixed embryos (E11.5 and E13.5) in ScaleU2 for 6 monthsand performed three-dimensional imaging of the left half of each body

using a Nikon AZ-C1 system (Fig. 6a,b). At both E11.5 and E13.5,the developing cerebral cortex contained more green nuclei than the

S/G2/M phase green, respectively. A transgenic mouse line that almostubiquitously expresses mAG-hGem(1/110), #504, was used previously

to obtain in vivo information about proliferation patterns at various

embryonic stages27.We labeled the vasculature of a #504 mouse with Texas Redlabeled

lectin. After fixation, the hippocampus was dissected out and treated

with ScaleA2 for 2 d. The cleared hippocampus was viewed with TPEFMat 920-nm excitation from its ventral surface (Fig. 5d) and a series of

perspective images aimed toward the dorsal side were taken (Fig. 5e).Green nuclei of PNSCs were localized exclusively in the SGZ in apparent

association with a network of blood vessels (Supplementary Video 4).We next examined the quantitative relationship between PNSCs of

the SGZ lineage and the vasculature. Multiple hippocampi were fixedand cleared by ScaleA2 and treated with 4,6-diamidino-2-phenylindole

(DAPI) to label nuclei. After performing TPEFM imaging on these prepa-rations, we made three-dimensional reconstructions that localized PNSC

nuclei and all nuclei in relation to blood vessels. The distances of the nucleito the nearest vessels were measured in three-dimensional space using the

RINZO algorithm (Fig. 5f), which we developed for this purpose.We analyzed 96 PNSC nuclei, out of 1,381 DAPI-positive nuclei in

the SGZ (Fig. 5g,h). Of the PNSC nuclei, 37% (35 of 96) were situatedwithin 10 mm of blood vessel surfaces. In contrast, only 22% of SGZ

nuclei (298 of 1,381) were within 10 m of blood vessels; these nucleimostly belonged to endothelial cells and pericytes, as well as PNSCs.

The average distance of PNSC nuclei to blood vessels (16.38 12.10 mm)was significantly closer (P< 0.05) than the average for all SGZ cell nuclei

(20.34 11.48mm). Because these analyses involved automatic process-ing (except for manual tagging of PNSC nuclei) and were performed

comprehensively in three-dimensional space, we conclude that PNSCnuclei are closely associated with blood vessels in the mouse SGZ.

Figure 6 Three-dimensional reconstructions of Fucci transgenic mouse

embryos treated with ScaleU2. (ac) Green and red signals are derived from

the Fucci-S/G2/M marker and Fucci-G1/G0 marker, respectively. Transgenic

mouse #596/#504 embryos (E11.5 and E13.5) were fixed with 4% PFA/

PBS and then incubated in ScaleU2 for 6 months. The right halves of their

bodies (heads) were imaged using macro-zoom LSCM (AZ-C1) equipped with

a 2 objective lens (AZ-PlanFluor, NA = 0.2, working distance = 45 mm).

The zstep size was 5 mm. We used 488-nm and 561-nm laser diodes.

Shown are maximum intensity projection (MIP) images at E11.5 (a) and

E13.5 (b). A confocal image of the region indicated by a white box in the

MIP image (b) is shown in c. (di) Immunohistochemical localization of

Nestin (df) or PSA-NCAM (gi) on sections of the posterior end of the

diencephalon of an E13.5 #504 transgenic embryo producing mAG-

hGem(1/110). The immunostaining and mAG-hGem(1/110) signals are

shown in white and green, respectively. High-magnification images of

the regions indicated by yellow boxes in e and h are shown in f and i,

respectively. IC, inferior colliculus; V, ventricle. Scale bars represent

1 mm (ac) and 100 mm (di).

E13.5

baE11.5

c

Fucci-S/G2/M

Fucci-S/G2/M

Fucci-S/G2/M

Fucci-G1

NestinV

ICVIC SC

PSA-NCAMV

ICV

IC SC

E13.5

E13.5

d e

g h i

f

E13.5

GluR1 Synaptophysin Merge

YFP PSA-NCAM Merge

SGZ

GCL

Hilus

SGZ

GCL

Hilus

Fixed

Scaled

restored

Fixed

Scaled

restored

Fixed

a b c

d e f

g h i

j k l

Fixed

Figure 7 Immunohistochemistry on sections restored from ScaleA2.

(af) A brain sample from the thy1-YFPmouse line H (7 weeks old) was

used. Sections of the dentate gyrus were prepared from a fixed sample (ac)

and a sample restored from ScaleA2 (df). Samples were stained with a

mouse monoclonal antibody to PSA-NCAM. The YFP fluorescence and

immunoreactivity for PSA-NCAM (with a secondary antibody conjugated to

Alexa Fluor 546) were visualized. (gl) A brain sample from wild-type mouse

(7 weeks old) was used. Sections of the CA3 region were prepared from a

fixed sample (gi) and a sample restored from ScaleA2 (jl). Samples were

stained with a rabbit polyclonal antibody to GluR1 and a mouse monoclonal

antibody to synaptophysin. These primary antibodies were visualized with

secondary antibodies conjugated to Alexa Fluor 488 and 546, respectively

(Molecular Probes). Scale bars represent 20 mm.


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Our results indicate that ScaleA2 is superior to other described

clearing agents in key properties necessary for performing detailedthree-dimensional brain reconstructions at both fine scale and broad

perspective (Supplementary Table 1). Unlike organic solventbasedreagents, Scale allows signal preservation of fluorescent proteins. Scale

is also superior to other aqueous reagents, including FocusClear, becauseit is inexpensive and its formula is simple and public, and to 60% sucrose,

which allows only modest transparency. Thus, a large quantity of Scalecan be used for clearing large tissue samples not only from rodent brains

and embryos, but, we expect, from primate28 and human biopsy samples.Furthermore, researchers can modify the reagents composition accord-

ing to the nature of the samples to be cleared. For example, the concen-tration of glycerol should be increased to preserve tissue volume and

integrity, and the concentration of urea can be reduced for brain samplesfrom fish and flies (data not shown). The drawbacks of ScaleA2 include

the long time periods required for clearance and the fragility of cleared

samples, but these problems may be largely solved by modifying reagentcomposition to ScaleB4 and ScaleU2, respectively.

Scale applications for brain development and function

Recent progress in gene targeting and genetic labeling methods has

revealed the roles of specific genes in regulating axon guidance duringthe formation of topographic projections. The Scale method will facili-tate the analysis of wild-type projection and screening of mutant mice

to detect those with aberrant axon projections. Moreover, the high reso-lution of Scale may allow mechanistic insights into the nature of such

defects. To follow up on our callosal findings, one could label axonswith different colors at different embryonic days to survey the develop-

mental structure of the corpus callosum29. The Scale technique can alsobe applied effectively to chemically stained samples, as we found with

transcardial perfusion of Texas Redlectin to label blood vessels beforefixation or in the future with in vivo labeling of astrocytes with Texas

Redhydrazide, a paraformaldehyde-fixable analog of SR101 (ref. 30),and neuronal tracing with NeuroVue dyes in fixed brain tissue31.

Further applications of the Scale system could involve studies of neu-ronal function. We labeled and reconstructed the proliferating NSC

niche in the SGZ, which was made possible by Scale clearance of thehippocampus. It will also be useful to obtain projection images of Scaled

brain samples using low-NA objective lenses to allow quantificationof the spatial expression of neural activityregulated immediate early

genes during behavior of transgenic mice in which the expression ofGFP is controlled by the promoter of c-fos32 or Arc33. Scale will thusbe valuable for the high-resolution immediate early gene mapping of

behavior in intact large-scale brain networks.

Scale applications for connectomics in intact brain

Scale should be compatible with most light microscopy systems. In par-

ticular, we suggest the utility of Scale-treated samples for light sheet

ultramicroscopy, a technique that can gather three-dimensional imagedata quickly13,14,34,35. In addition, in combination with Scale, it may bepossible to observe larger animals via fluorescent protein tomography

(FPT)36 using proteins that fluoresce at shorter wavelengths. Similarly, theScale technique should be applicable to all organs. Very few techniques

for three-dimensional reconstruction using light microscopy can pen-etrate tissue blocks thicker than 1 mm. In contrast, most tomographic

techniques, including optical projection tomography37, FPT36, computertomography and positron emission tomography, as well as magnetic

resonance imaging, are capable of analyzing structural and quantitativefeatures in a much larger mass of tissue, such as the whole body.

Among the emerging approaches for brain connectomics that aimat detailed three-dimensional reconstructions of brain structures, the

diencephalon, suggesting that cells in the diencephalon undergo cell-

cycle exit for differentiation before those in the cerebrum. Althoughneurogenesis in the diencephalon appeared to have reached completion

by E13.5, there was a region rich in green nuclei at the posterior endof the diencephalon (inferior colliculus) (Fig. 6b). Local cell prolifera-

tion was also evident in a sectional image along the midline (Fig. 6c).The green nucleuscontaining cells were immunostained for Nestin

(Fig. 6d

f), but not for PSA-NCAM (

Fig. 6g

i), which verified that theregion was still mitotic. Thus, the ScaleU2 technique applied to Fucci

transgenic mice provides a comprehensive perspective of proliferationversus differentiation in the developing brain.

ScaleA2 takes days or weeks to clear large specimens; ScaleU2 takesweeks or months. However, we found that the clearing process could

be accelerated by a transient tissue expansion induced by 8 M urea. Weexploited this observation to devise a speedy protocol that incorporates

a pulse of 8 M urea. In this protocol, fixed biological samples are incu-bated in ScaleA2 for 2 d, followed by ScaleB4 containing 8 M urea and

0.1% Triton X-100 for 2 d, and finally a ScaleA2 or ScaleU2 solution.As an added benefit, treating with ScaleB4 effectively depletes biologi-

cal samples of background signals. The pH of a ScaleB4 solution is 8.7;the stability of fluorescent proteins in 8 M urea (pH 8.7) was verified

(Supplementary Fig. 4a).It was not initially clear whether Scale treatment had an irreversible

effect on tissue architecture. We investigated whether a ScaleA2-treatedbrain sample could be restored to its original state by simply washing

with PBS. We split a fixed brain of the YFP-H mouse into two halves.The right half was kept in PBS while the left was cleared thoroughly with

ScaleA2 for 3 weeks and then washed in 20 volumes of PBS. After twosequential washes for 15 min each, the sample shrank to the original size

and became turbid. The two halves were embedded in optimal cuttingtemperature (OCT) compound, from which 30-mm-thick dentate gyrus

sections were prepared for histology. The patterns of immunostainingfor PSA-NCAM, as well as the cellular structures delineated with YFP

fluorescence, were very similar between the two samples ( Fig. 7af).

Next, we examined subcellular structures in the CA3 region using a wild-type mouse brain. When immunostained for the pre- and postsynapticmarkers Synaptophysin and GluR1, similar colocalization signals were

observed in sections from the two halves (Fig. 7gl). Similar results wereobtained with immunostaining for PSA-NCAM/BLBP (brain lipid bind-

ing protein) or VGLUT1 (vesicular glutamate transporter 1) using twoneighboring sections from a GFP-M mouse brain (Supplementary Figs. 8

and 9). It is therefore easy to restore a cleared specimen to its originalstate in a manner that is compatible with immunohistochemistry and

techniques such as array tomography2.

DISCUSSION

Comparison of Scale with other tissue-clearing techniques

With the Scale system, we present a simple, but effective, technique for

clearing mammalian brain tissue that has the potential to address ques-tions in brain structure and function at unprecedented spatial detail andscale. Our results demonstrate the utility of Scale for reconstruction of

neuronal populations and projections, in which labeled cells at subcel-lular detail can be viewed in three-dimensional networks at millimeter

scales. As Scale is part of an emerging area of chemical technology forimaging, we evaluated the strength and limitations of Scale, compared

its performance with that of other described clearing reagents and devel-oped solutions to extend its range of utility. Here we discuss its potential

roles among the emerging connectomics efforts to map the brain and itsconstituent structures. Given its simplicity and stability, we suggest that

Scale will popularize high-resolution three-dimensional reconstructionsin mammalian brain and other tissues, organs and animals.


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Scale technique will likely provide a simple, inexpensive complement

to array tomography and serial section electron microscopy efforts byenlarging the volume and depth of three-dimensional reconstructions

that can be achieved using light microscopy data. In this way, the Scalesystem should help to bridge the imaging gap38 between the size of

specimens that can be visualized with light microscopy versus otherbrain imaging techniques. The reversibility of Scale for retrospective

immunohistochemistry will enable zooming out to observe neuro-nal circuits in a global three-dimensional reconstruction, followed by

zooming in to image specific synaptic structures at both light andelectron microscopy scales. Together with advances in microscopy and

fluorescent proteins, Scale will contribute to the discovery of new bio-logical principles in connectomics and circuit genetics3941.

METHODS

Methods and any associated references are available in the online ver-

sion of the paper at http://www.nature.com/natureneuroscience/.

Note: Supplementary information is available on the Nature Neuroscience website.

ACKNOWLEDGMENTSWe thank H. Sakurai, H. Otsuka and M. Hirano for general assistance, F. Ishidate,

B. Zimmermann, R. Wolleschensky, Y. Watanabe, E. Nakasho, H. Kimura, T. Tajimaand S. Horie for help with acquiring and analyzing images, RIKEN BSI-OlympusCollaboration Center for technical support, Y. Yoshihara (RIKEN), M. Yamaguchiand K. Mori (The University of Tokyo) for the Nestin promoterGFP transgenicmice, J.R. Sanes (Harvard) for the YFP-H and GFP-M lines, E. Takahashi(RIKEN) for helpful advice on transgenic mice, S. J. Smith (Stanford) and J.W.Lichtman (Harvard) for helpful advice on tissue clearing, and D. Mou (Harvard),A. Govindarajan, K. Rockland and S. Tonegawa (Massachusetts Institute ofTechnology), A. Moore and C. Yokoyama (RIKEN) for critical comments. Thiswork was partly supported by grants from Japan Ministry of Education, Culture,Sports, Science and TechnologyGrant-in-Aid for Scientific Research on PriorityAreas and the Human Frontier Science Program.

AUTHOR CONTRIBUTIONSH.H. and A.M. conceived and designed the study. H.H. performed all theexperiments and analyzed the data. H. Kurokawa devised the algorithms andanalyzed the data. H. Kawano constructed the TPEFM system. R.A. performedin vitro experiments using fluorescent proteins. T.S. designed and performedthe experiments that imaged callosal connections. H.N. refined the algorithms.K.F. contributed to data analysis. A.S.-S. performed the experiments using Fuccitransgenic mouse embryos. A.M. supervised the project and wrote the manuscriptwith the help of H.H.

COMPETING FINANCIAL INTERESTSThe authors declare competing financial interests: details accompany the full-textHTML version of the paper at http://www.nature.com/natureneuroscience/.

Published online at http://www.nature.com/natureneuroscience/.

Reprints and permissions information is available online at http://www.nature.com/

reprints/index.html.

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gimmick. Neuron18, 351357 (1997).
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nature neuroscience advance online publication :10.1038/. 2928

Image acquisition using LSCM. In the experiment appearing in Figure 3hk,

brain samples of the YFP-H line were imaged using a ZEISS LSM710-NLO

system equipped with a 20 dipping objective (W-PlanApochromat, NA = 1.0,

working distance = 2.0 mm) and zdrive. The zstep size was 3.0 mm. YFP was

excited with a 514.5-nm Argon laser. The laser power was constant during image

acquisition; the excitation intensity at the level of the specimen was 2 mW. YFP

fluorescence was descanned and collected confocally. The size of the confocal

aperture was ~2 Airy disks (Supplementary Fig. 6b).

Image acquisition using macro zoom LSCM. Brain samples containing EYFP-

positive commissural connections were cleared with ScaleA2 and trimmed with

a coronal cut at the midbrain and positioned on a cover glass with the ante-

rior part uppermost. Transgenic mouse #596/#504 embryos (E11.5 and E13.5)

were cleared with ScaleU2. These samples were imaged using a Nikon macro

zoom confocal microscopy system (AZ-C1) equipped with a 1 objective lens

(AZ-PlanApo, NA = 0.1, working distance = 35 mm) or a 2 objective lens

(AZ-PlanFluor, NA = 0.2, working distance = 45 mm). EYFP and mAG were

excited with a 488-nm laser diode. mKO2 was excited with a 561-nm laser diode.

Pre-processing of images. Multiple xy images were tiled using the stitch func-

tion of a commercial program (Multi-Area Time-Lapse View of FV10-ASW,

version 2.0c) at each zposition. The correlation function was used to compute

the xy positions in the overlapping regions. All tiled xy images were processed

for shading correction by the custom-written filtering program JINARASHI.This program compensates signal intensity at the periphery of each image.

We developed JINARASHI using the Open CV Library (http://opencvlibrary.

sourceforge.net). JINARASHI is written in the C/C++ language and is available

on our website (http://cfds.brain.riken.jp/). JINARASHI corrects signal gradi-

ents on the basis of the background intensity distribution. Shading-corrected

images were stacked to achieve three-dimensional reconstruction using the

volume-rendering function of the commercially available Volocity version 5.3

(Improvision/PerkinElmer). Volume-rendered images were displayed using

fluorescence mode (three-dimensional opacity) and exported as AVI images.

Measurement of distances. Nuclei emitting mAG-hGem(1/110) green fluores-

cence or those stained with DAPI in the SGZ regions were tagged manually. In

contrast, blood vessels were automatically segmented using a custom-written

program, Frame Level Threshold, which converts the signal into binary. This

program is written in the C/C++ language and is available on our website (http://cfds.brain.riken.jp/). The distance from each nucleus to the nearest blood ves-

sel surface was measured using a custom-written program, RINZO. Different

parameters were set depending on the voxel shape, that is, the ratio of the length

of an x ory edge to that of a zedge. RINZO is written in Java and is available on

our website (http://cfds.brain.riken.jp/). Data were pooled from six SGZ regions.

Transmission measurement. Transmission curves were acquired using a U3310

spectrophotometer (Hitachi). Fixed brain slices (3 mm thick) were incubated

in PBS, a 60% sucrose solution, FocusClear or a Sca leA2 solution. The slices

equilibrated with sucrose or FocusClear shrank slightly, whereas the slices

treated with ScaleA2 expanded slightly. After incubation, slices with a thick-

ness of exactly 1.5 mm were generated from the equilibrated samples using

a tissue slicer. These 1.5-mm-thick slices each were mounted in a cuvette for

measurement of transmission.

Immunohistochemistry. Adult mice (7-9 weeks old) were deeply anesthetized

with pentobarbital (Nembutal) and transcardially perfused with 4% PFA/PBS.

After postfixation in 4% PFA/PBS and cryo-protection in 20% sucrose/PBS,

brains were embedded in OCT compound (Sakura). Restored brain samples

from ScaleA2 treatment were also embedded in OCT compound. Sagittal sec-

tions (35 mm thick) were cut with a cryostat. Sections were permeabilized/

blocked for 30 min in 0.1% Triton X-100 (wt/vol)/10% horse serum (vol/vol)/

PBS and then processed by free-floating immunohistochemistry. For double

staining, primary antibodies from different species were incubated simultane-

ously, followed by incubation with secondary antibodies.

For primary antibodies, we used mouse monoclonal antibody to PSA-NCAM

(Millipore), rabbit polyclonal antibody to GluR1 (Millipore) and mouse mono-

clonal antibody to synaptophysin (Sigma). For secondary antibodies, we used

ONLINE METHODS

Scale solutions. Stock solutions containing high concentrations of urea (Wako

Chemicals) or Triton X-100 (Wako Chemicals) were prepared. Sca le solutions

were made by mixing the stock solutions and glycerol (Sigma). The f inal con-

centrations of urea, glycerol and Triton X-100 were adjusted by diluting the

mixed solutions with water.

Sample preparation. Neonatal and adult mice (2-13 weeks old) were deeply

anesthetized with pentobarbital (Nembutal) and killed by transcardial perfu-

sion with 4% PFA/PBS (wt/vol). The whole brains were taken out and subjected

to postfixation in 4% PFA/PBS at 4 C for 10 h and cryo-protection in 20%

sucrose/PBS (wt/vol) at 4 C for 24 h. For observation of the entire hippocampal

formation, the hippocampi were excised. Brains or hippocampal samples were

embedded in OCT compound and frozen. They were thawed in PBS and fixed

again with 4% PFA/PBS for 20 min at 25 C. Next, the samples were cleared in a

ScaleA2 solution (20 ml per 0.5 g tissue) at 4 C for >2 d. Mouse embryos (E13.5)

were transcardially perfused with 4% PFA/PBS, postfixed for 2 h and cleared

with ScaleA2 for 2 weeks. Alternatively, mouse embryos (E11.5 and E13.5) were

fixed with 4% PFA/PBS for 2 h and cleared with Sca leU2 for 6 months. For

speedy processing, fixed samples were initially cleared with ScaleA2 or ScaleU2

for 2 d, with ScaleB4 for 2 d, and then with ScaleA2 or ScaleU2 again. The experi-

mental procedures and housing conditions for animals were approved by the

Institutes Animal Experiments Committee of RIKENand all of the animals werecared for and treated humanely in accordance with the Institutional Guidelines

for Experiments using animals.

Imaging of fixed HeLa cells. HeLa cells grown on a 35-mm glass-bottom dish

were transiently transfected with cDNA for EGFP using Lipofectin (Invitrogen).

After fixation with 4% PFA for 10 min, cells were kept in Hanks Balanced Salt

Solution containing 15 mM HEPES-NaOH (pH 7.4) and time-lapse imaged

using an inverted microscope (IX81, Olympus) equipped with a standard 75-W

xenon lamp, a 40 objective lens (UplanFlN Oil, NA = 1.30) and a cooled CCD

camera (iXon EM+, Andor Technology). EGFP signals were obtained using

an excitation filter (470 10 nm), an emission filter (517.5 22.5 nm) and a

dichroic mirror DM485. The system was operated using MetaMorph 7.6 soft-

ware (Molecular Devices).

In utero

electroporation. After anesthesia with sodium pentobarbital, preg-nant mice at E15.5 were subjected to abdominal incision, and all the uterine

horns were exposed onto PBS-moistened cotton gauze. Embryos were visualized

through the uterine wall using a f lexible fiber cable, and plasmid DNAs mixed

with the non-toxic dye Fast Green were injected into the lateral ventricle through

a pulled glass capillary. A pair of platinum electrodes was applied to the uterus,

and a series of square-wave current pulses (38 V, 50 ms) was delivered five times

at 1-s intervals using a pulse generator. Uterine horns were repositioned into the

abdominal cavity, and the abdominal wall and skin were sutured.

Image acquisition using TPEFM. The light source used for TPEFM was a

femtosecond-pulsed Ti:sapphire laser. When YFP- or GFP-expressing neurons

were imaged, an excitation wavelength of 920 nm was used. Emitted light was

collected by an external non-descanned detector (Supplementary Fig. 6a). In

the experiments shown in Figure 3ac,lo and Supplementary Figure 7ae,

multiple neighboring regions in the brain were imaged using a TPEFM system(Olympus FV1000MPE + Coherent Chameleon Ultra-II + PreComp) equipped

with a motorized xy stage module in addition to a motorized focus module

(Z-drive). Adjacent regions overlapped by 10% to allow precise alignment.

Lenses used included a 40 dipping objective (LUMPLFLN40XW, NA = 0.8,

working distance = 3.3 mm), a 25 dipping objective (XLPLN25XWMP, NA =

1.05, working distance = 2.0 mm) or a custom-designed 25 objective lens (NA =

1.0, working distance = 4.0 mm). The brightness compensation function in the

zdirection was used to change the detector sensitivity and laser power. In the

experiment represented in Figure 3dg, single regions were imaged using a

ZEISS LSM710-NLO system (+ Spectral Physics MaiTai HP DeepSee) equipped

with a 20 dipping objective (W-PlanApochromat, NA = 1.0, working distance =

2.0 mm) and zdrive. The laser power was constant during image acquisition;

the excitation intensity at the level of the specimen was 6 mW.
http://cfds.brain.riken.jp/http://cfds.brain.riken.jp/http://cfds.brain.riken.jp/http://cfds.brain.riken.jp/http://cfds.brain.riken.jp/http://cfds.brain.riken.jp/


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:10.1038/.2928 advance online publ ication nature neuroscience

Transmitted-light bright-field (transmission) images were acquired with oblique

illumination and a reduced condenser aperture42, which can enhance struc-

tural differences in inherent absorption in the brain slices. Contrast was further

enhanced in the final images by electronic image processing. Fluorescence (YFP)

images were acquired using an excitation filter (480 20 nm) and an emission

filter (510-nm long pass). The outlines of the slices and several internal struc-

tures in the images were traced. The extent of the linear expansion (E) was calcu-

lated by comparing the distances across highlighted structures between 0-d and

5-d images. At 5 d, the image was reduced in size by 1 1/E for superposition.

Statistical analysis. The statistical analysis for Figure 5g,h was performed using

the Mann-WhitneyUtest. Difference was considered to be significant when

P< 0.05. The data described in the text represent means s.d.

42. Keller, H. E. Contrast enhancement in light microscopy. Curr. Protoc. Cytom. 2.1.1

2.1.11. (2001).

goat antibody to rabbit IgG conjugated to Alexa Fluor 546 (Molecular Probes),

goat antibody to mouse IgG conjugated to Alexa Fluor 546 (Molecular Probes), goat

antibody to rabbit IgG conjugated to Alexa Fluor 488 (Molecular Probes) and goat

antibody to rat IgG conjugated to Alexa Fluor 633 (Molecular Probes).

Observation of macroscopic structures in brain slices. Fixed brains were frozen

in OCT compound, thawed in PBS and fixed again with 4% PFA/PBS for 20 min

at 25 C. Then, coronal slices (1 mm thick) were cut with a razor blade from the

brain sample placed in the Mouse Brain Slicer (MB-A1-C, Muromachi Kikai).

Slices containing the hippocampus and the striatum were selected. These slices

were cut near the midline and the right halves were incubated in ScaleA2 solu-

tion at 4 C for 5 d, while the left halves were kept in PBS. Intermittently, these

slices were placed on a coverslip with a patterned background and imaged using

a Leica fluorescence stereomicroscope (MZ16F) equipped with a 1 objective

lens (PLANAPO, NA = 0.141) and a cooled CCD camera (DP30, Olympus).

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