A Method for 3D Immunostaining and Optical Imaging of
the Mouse Brain Demonstrated in Neural Progenitor
Cells
Jacqueline A. Gleave1,2, Jason P. Lerch1,3, R. Mark Henkelman1,3, Brian J. Nieman1,3*
1 Mouse Imaging Centre, Hospital for Sick Children, Toronto, Ontario, Canada, 2 Department of Biological Sciences, University of Toronto, Toronto, Ontario, Canada,
3 Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada

Abstract
It is important to understand changes in cell distribution that occur as a part of disease progression. This is typicallyachieved using standard sectioning and immunostaining, however, many structures and cell distribution patterns are notreadily appreciated in two-dimensions, including the distribution of neural stem and progenitor cells in the mouseforebrain. Three-dimensional immunostaining in the mouse brain has been hampered by poor penetration. For this reason,
we have developed a method that allows for entire hemispheres of the mouse brain to be stained using commerciallyavailable antibodies. Brains stained for glial fibrillary acidic protein, doublecortin and nestin were imaged in threedimensions using optical projection tomography and serial two-photon tomography. This staining method is simple, using
a combination of heat, time and specimen preparation procedures readily available, so that it can be easily implemented
without the need for specialized equipment, making it accessible to most laboratories.
Citation: Gleave JA, Lerch JP, Henkelman RM, Nieman BJ (2013) A Method for 3D Immunostaining and Optical Imaging of the Mouse Brain Demonstrated in
Neural Progenitor Cells. PLoS ONE 8(8): e72039. doi:10.1371/journal.pone.0072039
Editor: Chin-Tu Chen, The University of Chicago, United States of America
Received April 6, 2013; Accepted July 3, 2013; Published August 6, 2013
Copyright: ! 2013 Gleave et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Ontario Institute for Cancer Research through funding provided by the Government of Ontario. The funders had norole in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: bjnieman@mouseimaging.ca

Introduction

available and it is impractical and expensive to generate such micefor studies where multiple markers are necessary simultaneously or
where the breeding is already complicated due to the diseasemodel being investigated.
Adaptation of staining methods with commercial antibodies, as
used for traditional 2D immunohistochemistry, would provide
much more flexibility to 3D optical imaging methods, enhance theimpact and convenience of these tools, and enable routine analysis
of cell and gene product distributions in 3D. Although antibody
staining in 3D samples has been successful in some tissues [13,14],
it has posed challenges in the mouse brain due to low penetrationof the antibodies, preventing the staining of cells deeper than a few
hundred microns [15]. Therefore, we developed a straightforward
antibody staining method that allows for penetration of antibodies
in intact mouse brain samples. This method is flexible, can be usedwith a number of antibodies, allowing for the spatial distribution ofmultiple cell types to be assessed simultaneously, and is applicable
to any 3D optical imaging modality. The staining method itself issimple and easy to apply, using a combination of heat, time, andspecimen handling procedures available in most laboratories toincrease antibody penetration into the mouse brain. Here we
carefully evaluate the quality of the staining in mouse brain
samples, focusing on neural progenitor cell distribution, andprovide demonstrations of its potential and limitations for 3D
visualizations.

The spatial distribution of various cell types or proteins isfundamental to understanding normal and pathological processes
in the brain. Many studies use mouse models to probe theimportance of certain cells or proteins and then rely on histologicalsectioning and antibody staining to generate representative twodimensional (2D) sections. However, many structures or cell
distributions, such as migrating neural progenitor cells, vasculature, and branching axonal connections, are not readily appreciated in 2D. While alignment of serially-stained sections is apossible workaround for this problem, it is difficult, laborious andimpractical for routine use. Furthermore, comparison between
control and experimental groups in a study routinely requires
cutting and identification of equivalent sections in multiple
specimens, a subjective process that can be difficult even in simple
cases.
For these and other reasons, several optical imaging methods
have been developed that enable imaging of the mouse brain
directly in three-dimensions (3D) [1–4]. Examples include opticalprojection tomography (OPT) [5,6], light sheet fluorescent
microscopy [7–9], blockface imaging [10,11], and serial twophoton tomography [4]. With many of these tools, cell types orgene products of critical interest can be visualized using transgenic
optical markers, such as fluorescent proteins, under the control ofappropriate promoters. New methods of optically clearing
specimens will further expand the application of these techniques[12]. However, the appropriate transgenic mouse is not always

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3D Immunostaining and Imaging in the Mouse Brain

Materials and Methods

were incubated at 37uC for 48 hours in primary antibody diluted
in 5% normal goat serum (Jackson ImmunoResearch, PA, USA),
5% dimethylsulfoxide (DMSO, Fisher Scientific, Ontario, Canada), and 0.01% Triton X-100 (Bioshop Canada, Ontario,
Canada). The samples were then washed at 37uC for 48 hours
in 5% serum, 0.01% Triton X-100, replacing the solution once
after 24 hours. Following this, the samples were incubated for
72 hours in secondary antibody diluted in 5% serum, 5% DMSO,
and 0.01% Triton X-100. Finally, the samples were washed at
37uC for 48 hours in 0.01% Triton X-100, replacing the solution
once after 24 hours.
Staining of single hemisphere brain samples using the described
protocol for 3D imaging included the following: doublecortin(n = 4), GFAP (n = 4), nestin (n = 3), double stain of doublecortin
and nestin (n = 1). Doublecortin was also used to stain a full mousebrain excluding the cerebellum (n = 2).
Control samples were generated using pre-absorbed primary
antibodies when possible by incubating the primary antibody witha blocking peptide for 30 minutes at room temperature prior tostaining. If no peptide for the primary antibody was available, thestaining procedure was performed in the control except that the
primary antibody was omitted during the appropriate step.
Staining was performed with the following antibodies: rabbit
anti-doublecortin (Abcam), goat anti-doublecortin and peptide(Santa Cruz Biotechnology) rabbit anti-GFAP (Dako), chicken
anti-nestin (Neuromics), goat anti-rabbit alexafluor 546 (Invitrogen), goat anti-rabbit alexafluor 488 (Invitrogen), goat anti-rabbit
alexafluor 647 (Invitrogen), goat anti-chicken alexafluor 546(Invitrogen), goat anti-chicken alexafluor 488 (Invitrogen), donkey
anti-goat alexafluor 546 (Invitrogen), donkey anti-goat alexafluor
488 (Invitrogen).

Animals
All animal experiments were approved by the animal care
committee for the Toronto Centre for Phenogenomics.

Perfusion
Eight-week old male wildtype C57Bl6/J (Toronto Centre forPhenogenomics, in-house breeding, Toronto, Ontario, Canada)
were anesthetized with an intraperitoneal injection of 150 mg/kg
ketamine and 10 mg/kg xylazine.
1% PFA perfusion. Anesthetized mice were perfused intracardially with 15 ml phosphate buffered saline (PBS, Wisent
Bioproducts, Quebec, Canada) containing 10 U/ml heparinfollowed by 15 ml of 1% PFA. The brains were removed fromthe skull and soaked for 2 hours in 1% PFA and subsequently
washed with PBS.
4% PFA perfusion. Anesthetized mice were perfused intracardially with 30 ml PBS containing 10 U/ml heparin followed by
30 ml of 4% PFA. The brains were soaked in the skull overnight at
4uC. The brains were washed in PBS and removed from the skulls
the following day.

Diffusion of 150 kDa FITC-dextran
Samples approximately 4 mm in each dimension were cut using
an adult mouse brain matrix (Kent Scientific Corp, Torrington,CT) and then incubated with 150 kDa FITC-dextran (Sigma,
Ontario, Canada) for 5, 10, 24, or 48 hours at 4uC or 37uC. The
samples were then sectioned into 50 mm sections on a vibratome(Leica, Germany), visualized using an inverted fluorescent
microscope (Leica, Germany), and digitally captured using acooled, CCD camera (Qimaging, BC, Canada). The microscope
images were stitched together to visualize the entire section in asingle image, and intensity curves representing diffusion of FITCdextran into the sample were obtained by mapping the intensity
along the line normal to the specimen edge and toward the centreof the section. Approximately 20 such linear intensities wereobtained for each section. Subsequently, the linear distance alongeach curve was normalized by the square root of the incubation
time. The intensity of each curve was also normalized, using athird-order polynomial fit to estimate the amplitude at the origin.
After both the distance and intensity normalizations, equivalent
curves were averaged together for visualization and plotted with90% confidence intervals.

2D immunostaining
Unstained or previously 3D-stained mouse brains were soakedin 30% sucrose to prepare them for cryosectioning. 10 mm
cryosections were cut and the OCT was removed in a PBS wash.
Antigen retrieval was performed at 90uC for 20 minutes followedby allowing the slides to cool to room temperature. The slides were
washed 3x with PBS and once with 0.1% Triton X-100 and
subsequently blocked with 5% normal goat serum. The slides wereincubated with primary antibody (listed above) overnight at 4uC in
1% normal goat serum and 1% Triton X-100. The slides werethen washed 5 minutes in PBS three times followed by a 1 hour
incubation at room temperature in secondary antibody diluted in
1% normal goat serum, 0.1% Triton X-100. The slides were
washed for 5 minutes in PBS three times followed a 5 minute wash
in dH2O. Finally, the slides were rinsed with dH2O and mountedwith Vectashield hard-set with DAPI mounting medium (Vector
Labs, Ontario, Canada).
To test the primary and secondary staining steps of the 3D stain,
slides from the previously stained brains were incubated witheither both primary and secondary antibodies or secondary
antibody only. All 3D staining used antibodies conjugated toalexafluor 546 and the follow-up restains used secondary
antibodies conjugated to alexafluor 488.

3D antibody staining of adult mouse brains
1% PFA perfused adult mouse brains were removed from theskull and then divided by removing the cerebellum and separating
the hemispheres if desired. The samples were immediately
dehydrated in a gradient of methanol solutions to 100% methanol
over the course of one day. Immediate dehydration is important topreserve the cellular morphology of the lightly-fixed brain. The
samples then underwent freeze/thaw (one hour at –80uC/onehour at room temperature) four times. The samples wererehydrated in a gradient of methanol solutions to PBS over thecourse of one day. The samples were mildly digested for 5 minutes
using 10 mg/ml of Proteinase K (Promega, WI, USA) and thenthoroughly washed to remove any residual Proteinase K. Antigen
retrieval was performed using a Histos5 histology microwave(Milestone, MI, USA) in 0.01 M sodium citrate buffer pH 6 on an
antigen retrieval program (0–90uC 11min, 90–98uC 3min, 98uC
10min). The samples were cooled to RT and subsequently washedin PBS to remove excess buffer. From this point, all incubations
were performed in the Histos5 histology microwave. The samples
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Sample preparation for imaging
Optical projection tomography (OPT). Samples wereembedded in a 1% low melting point agarose (Fisher Scientific,
Ontario, Canada) in a cylindrical mold. The agar was allowed tosolidify and the sample was dehydrated through a gradient of
ethanol solutions. The sample was cleared in benzyl benzoate(Sigma, Ontario, Canada):benzyl alcohol (Fisher Scientific,
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3D Immunostaining and Imaging in the Mouse Brain

Ontario, Canada) (2:1) over the course of 3 days. The cleared
sample was imaged using OPT.
Serial two-photon tomography. After staining of samples
fixed in 1%PFA as described above, samples were post-fixed
overnight at 4uC in 4% PFA. This made the samples stiffer so thatthey could be cut more cleanly using the integrated vibratome ofthe serial two-photon tomography system. The following day, thesamples were washed three times in PBS to remove any excess
PFA and then stored overnight in PBS at 4uC. The samples werethen embedded in 3% agarose (Bioshop Canada, Ontario,
Canada) made with phosphate buffer [4].

Results
A simple protocol allows increased diffusion ofmacromolecules into the mouse brain

We assayed diffusion of 150 kDa dextran FITC in ,4 mm
hemispheres of the mouse brain under various conditions in theaim of designing a protocol that would increase the penetration
depth for antibody staining (Figure 1). After completing a ‘‘stain’’
with the dextran FITC, the brain specimen was cut on a
vibratome and examined under a microscope. The standard
protocol from which we started performed the ‘‘stain’’ at 4uC, inwhich diffusion appeared hindered, producing a band ofconcentrated fluorescence and a sharp boundary. We found that
increasing the temperature to 37uC using a histology microwave
increased the dextran FITC penetration and produced afluorescence intensity profile more consistent with free diffusion.
Decreasing the concentration of the fixative used from 4%paraformaldehyde (PFA) to 1% PFA modestly increased the depthof penetration further. Incorporating pre-staining processing steps
that included freeze/thaw cycles and a short proteinase K
digestion [15] increased penetration sufficiently to ‘‘stain’’ thehalf-brain sample in a reasonable time frame. We incorporated
these steps into a 3D antibody staining protocol for the adultmouse brain that proceeded through the following steps: (1)1%PFA fixation; (2) dehydration, freeze-thaw, rehydration andbrief proteinase K digestion; (3) antigen retrieval (,10mins at
98uC, ,24mins total); (4) 48 hour soak in primary antibody(37uC); (5) 48 hour wash (37uC, change solution at 24 hours); (6)72 hour soak in secondary antibody (37uC); and (6) 48 hour wash
(37uC, change solution at 24 hours). When necessary, an optional

3D Optical Imaging
OPT. Images were acquired on a custom-built OPT scanner[16], the details of which will be published separately. 1200
projection views were taken over 360u. Images were acquired
using a l = 425615 nm excitation filter and a l = 473 nm longpass emission filter for autofluorescene imaging, l = 531620 nm
excitation filter and l = 593620 nm emission filter for imaging
alexafluor 546, and a l = 628620 nm excitation filter andl = 692620 nm emission filter for alexafluor 647 imaging with a102461024 matrix on a cooled CCD camera (Qimaging, BC,
Canada). The projections were reconstructed into the final 3D
image using SkyScan NRECON Software (Bruker, Belgium).
Serial two-photon tomography. Images were acquired
using a TissueCyte 1000 (TissueVision, Cambridge, MA, USA)
serial two-photon tomography system using l = 800 nm, 600 mW
laser (Coherent, CA, USA) and a single-edge dichroic beamsplitterwith l = 552 nm with an in-plane resolution of 1.37 mm and athrough-plane pixel resolution of 0.1 mm. Individual tiles werereconstructed in post-processing to generate the full 3D image.

Visualization
A non-uniformity correction [17] of the autofluroescent OPT
images was performed before display to correct for the apparently
brighter intensity at the edge of these images that results due tolight scatter and penetration. To approximate and remove theautofluorescence background from the stained image channel, ascale factor was computed by linear regression to match theautofluorescence channel (green) to the background of the stain(red). Subsequently, the scaled autoflurescence results weresubtracted from the stain. A similar correction was also performed
for the serial two-photon tomography images. For 3D display ofsome images, we applied a nonlinear low-pass filter to remove
isolated high-intensity pixels (as described in the results for DCX
and nestin staining). The isolated pixels were detected by
performing a high-pass filter, normalizing the resulting image tothe local standard deviation, and then thresholding to identify
outliers. Subtraction of the high-pass filtered image from theoriginal image where outliers were detected produced the filteredresult for display and analysis.

Density Maps
For GFAP serial two-photon tomography and histology images,
cell area density was quantified and mapped in 2D slices for
qualitative comparison. Images were blurred with a Gaussian filter
and then cells were detected based on a high local intensity using
both an intensity threshold and a local rank filter. Each identified
pixel cluster was then represented with an average pixel locationand taken to represent a single cell. An area density map was
generated on a downsampled grid. At each grid point, the area
density was computed by locating the eight nearest cell locations,and then using the average of their square distance to estimate theassociated area.
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Figure 1. Dextran-FITC diffusion in brain specimens. 50 mm
slices from a 4 mm hemisphere ‘‘stained’’ with dextran-FITC for
24 hours under different conditions as shown. Average one-dimensional intensity profiles from the edge of the tissue toward the center
across several samples are shown at lower right with 90% confidence
intervals. Samples processed for different lengths of time werecombined by scaling the linear diffusion distance by the square rootof time. The combination of increasing temperature, utilizing a 1% PFA
fixation, and processing with a freeze/thaw, and proteinase K digestion(+FT) resulted in the biggest improvement to penetration.
doi:10.1371/journal.pone.0072039.g001

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corpus callosum and mirrored the section obtained by traditional
methods in the forebrain, although in more caudal regions staining
was sparser and lighter than expected, particularly in the
hippocampus. In the case of DCX and nestin, but not GFAP,
we also observed high-intensity punctate fluorescence in the 3D
stain not representative of staining in the traditional sections. The
same punctate appearance can be replicated in a 2D-stained
section by mixing primary and secondary antibody in solution
prior to ‘‘staining’’, suggesting that the punctate voxels weobserved are likely due to unreacted primary after an incomplete
wash step. Lengthening the wash step or decreasing the primary
antibody concentration could reduce these artifacts, though at thecost of increased sample preparation times or incomplete staining.
As an alternative, to keep the sample preparation time short in 3D
imaging applications, these pixels/voxels can also be identified as
isolated high-intensity pixels/voxels and then be filtered from theimage on this basis.
We subsequently examined the 3D staining in more detail by restaining sections with traditional methods after first preparing
them with the 3D method in intact brain hemispheres. First,
subsets of sections were re-stained with a secondary antibody
conjugated to a different color fluorophore to visualize regions
where primary antibody had not been properly visualized bysecondary antibody. Red secondary antibodies were used for the
3D stain, and green secondary antibodies were used for the restain in individual sections. We observed that in the cases of DCX
and nestin, there was no primary antibody that remained unboundby secondary antibody, so that little or no green secondary could
be observed (Figure 2C, Figure 3A). On the other hand, a re-stain
with green secondary antibody for GFAP did show regions where
additional binding took place. In the forebrain, there was complete
overlap in the green staining and the red 3D stain (Figure 3A),
indicating that penetration of the secondary antibody wasadequate to show the complete cell distribution but that additional
secondary antibody binding sites were available. In more caudal
regions of the brain, there were some cells where the 3D staininglabeled GFAP-positive cells, but were only faintly visualized. The
re-stain in sections with green secondary antibody only showedmodest binding, suggesting that penetration of secondary anti-

Figure 2. Validation of DCX staining. A traditional 2D stain of DCX(A) shows a reference distribution of migrating neuroblasts (red). The
3D staining method in a brain hemisphere (B) shows a comparable
distribution (arrows, A and B), with the exception of some additionalpunctate fluorescence (arrowheads, B). Cryosections prepared from the
3D stained hemisphere were re-stained with another secondary
antibody (green; C) to highlight primary antibody left unbound bysecondary during the 3D stain; the primary antibody is bound bysecondary in the 3D staining method (arrows). In (D), an additional
cryosection from the same brain as (C) was re-stained with both primaryand secondary antibody to highlight antigen binding sites not occupied
by primary. Overlap of the 3D-stained and re-stained cells (arrowheads,
D), indicate that no DCX cells have been missed in the 3D-stained
images. LV = lateral ventricle, scalebars = 100 mm
doi:10.1371/journal.pone.0072039.g002

final step can be added with an additional 4%PFA fixation tofurther stiffen tissue for cutting of thin sections for 2D or 3D
imaging methods (i.e., variants of blockface imaging).

‘‘3D staining’’ in intact specimens produces similar resultsover much of the brain as traditional immunostaining
We tested the 3D staining method with three different
antibodies in single hemispheres from adult mouse brains andthen sectioned the brains for comparison with traditionally-stained
sections visualized with fluorescence microscopy. An example ofthese results is provided in Figure 2 for staining with doublecortin(DCX), which marks neuroblasts in the subgranular zone (SGZ)
and the subventricular zone (SVZ), from which they migrate along
the rostral migratory stream (RMS) to the olfactory bulbs [18]. We
also evaluated 3D staining for nestin and glial fibrillary acidic
protein (GFAP) (Figures 3 and 4 respectively). Nestin is a marker ofneuronal stem and precursor cells in the adult mouse brain [19]and GFAP stains both astrocytes and neural stem cells [20].
Overall, we observed a similar distribution of DCX-staining inspecimens prepared by the 3D staining method and thencryosectioned as we did in specimens prepared traditionally by
first cryosectioning and then staining (Figure 2a and b). Likewise,
3D nestin staining showed the expected distribution of nestinpositive cells surrounding the lateral ventricles. 3D GFAP staining
also showed comparable staining of white matter, such as the
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Figure 3. Validation of 3D nestin staining. Cryosections cut from astained brain hemisphere show nestin-positive cells (red; arrows, A).
Some additional punctate fluorescence was present (arrowheads, A).
The sections were re-probed with a secondary antibody (green; A) tohighlight primary antibody left unbound by secondary antibody. No
evidence of primary antibody left unbound by secondary antibody was
found. Sections were also re-stained with both primary and secondary
antibodies (B) to highlight primary-antibody antigen sites unoccupied.
There is overlap of the original 3D nestin stain with the 2D re-stain(arrowheads). LV = Lateral ventricle, scalebars = 100 mm.
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Figure 4. Validation of 3D GFAP staining. Cryosections from a the anterior (A, B) and posterior (C–F) of a GFAP-stained brain hemisphere areshown. Restain with another secondary antibody (green; A, C, D) highlights the presence of primary antibody left unbound by secondary antibody.
Restain with both primary and secondary antibody (green; B, E, F) highlights the presence of additional antigen sites initially unoccupied by primary.
Arrowheads highlight overlap of the re-stain and the original 3D stain. Arrows highlight cells that are only seen with the re-stain. In the anterior
region, there remains complete overlap between the 3D stain (red) and the re-stains (A, B). In the posterior potion of the brain, there is overlap of thesecondary antibody re-stain and the original 3D stain in the hippocampus (C) and posterior portion of the lateral ventricle (D). Re-stain with bothprimary and secondary antibody shows the presence of unoccupied primary antibody-antigen sites, including some cells not stained with the 3D
method in the hippocampus (E) and by the posterior lateral ventricle (F). LV = lateral ventricle, scalebars = 100 mm.
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Figure 5. 3D OPT imaging of DCX staining in the mouse brain. 3D OPT of a DCX-stained single hemisphere are shown as optical slices. DCX
staining is visible surrounding the lateral ventricle (A; i–iii) and in the subgranular zone of the dentate gyrus (A; iv). A cartoon shows the location of
the slices. The chains of DCX migrating neuroblasts along the lateral ventricle can be visualized on the ventricle surface in 3D (B). In (C), a maximum
intensity projection of a full brain shows cells migrating along the RMS to the olfactory bulbs (shaded green).
doi:10.1371/journal.pone.0072039.g005

antigen sites were left unoccupied by the original primary
antibody. For GFAP, the anterior region of the brain similarly
showed no unstained cells (Figure 4B). However, in more caudal
brain regions, the re-stain in sections with both primary and green
secondary showed that there were some areas where the primary
antibody penetration was incomplete, specifically in the hippocampus where some cells showed only green fluorescence from there-stain (Figure 2E and F). This indicates that, using theunmodified protocol we assayed, the GFAP primary antibody

bodies during the 3D stain was not limiting visualization of thsecells (Figure 4C and D).
Finally, another subset of sections cut from specimens stainedwith the 3D method were re-stained with both primary andsecondary antibody to visualize any regions left unstained by the
primary during the 3D staining procedure. In the case of DCX
and nestin, restained regions were observed, but found to overlap
with the 3D stain (Figure 2D, Figure 4B), indicating that cells had
been adequately labeled by primary but that some additional
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Figure 6. 3D OPT imaging of nestin staining in the mousebrain. 3D optical imaging with OPT was used to image a nestin-stained
brain hemisphere. Optical slices of the brain are shown and nestinpositive cells are visible surrounding the lateral ventricle. A cartoon
shows the location of the slices.
doi:10.1371/journal.pone.0072039.g006
Figure 7. 3D OPT imaging of GFAP staining in the mouse brain.
3D optical imaging with OPT was used to image a GFAP-stained brain
hemisphere. Optical slices of the brain are shown and GFAP-positive
cells are seen throughout the brain (A). A cartoon shows the location of
the slices. A 3D representation shows the distribution of GFAP staining
along the corpus callosum (B).
doi:10.1371/journal.pone.0072039.g007

was slower to penetrate the posterior region of the brain and thisresulted in poorer staining in parts of the hippocampus.

Antibody staining can be visualized in brain samples with
3D optical imaging
The key benefit of doing 3D staining is the ability to visualize
the distribution of the stained cells in a larger anatomical context.
This allows appreciation of cell distributions that are difficult toassess in 2D, such as, for example, the chains of DCX-positive
migrating neuroblasts. In Figure 5, optical slices from an OPT
image of a single hemisphere stained with DCX are shown with
autofluorescence in grayscale to show anatomy and a color overlay
showing DCX stain. DCX-positive cells are found in the expectedareas of the brain including the RMS, SVZ surrounding the lateral
ventricles, and the SGZ in the hippocampus (Figure 5A).
Projection of the DCX intensities onto a surface rendering of
the lateral ventricles shows the migrating chains of DCX-positive
cells on the lateral face (Figure 5B), consistent with expectations
from published work [18]. A maximum intensity projection of
DCX staining in a whole mouse brain (without the cerebellum)
shows the distribution of the stain in full anatomical context andallows the migrating chains from the SVZ of the lateral ventricles
along the RMS to the olfactory bulbs to be visualized (Figure 5C).
Similarly, optical slices of nestin- and GFAP-stained brains imaged
with OPT demonstrate the application of this method for otherantibodies (Figures 6 and 7). As is common practice for traditional
antibody staining in thin sections, a 3D control stain was also
performed for each stain to verify specificity by staining with eithera pre-absorbed primary antibody or a secondary antibody in theabsence of primary antibody.
The staining method as described above for OPT can be readily
applied to other 3D optical imaging modalities. To demonstrate
this, we imaged DCX-, nestin- and GFAP-stained samples using
serial two-photon tomography [4], a technique that combines
block face imaging with two-photon microscopy and increases the
pixel resolution to ,1 mm. This resolution is comparable to 2D
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histological sections. The distributions of DCX-, nestin-, and
GFAP-positive cells are found in the expected areas and mirror the
OPT results (Figures 8, 9, and 10 respectively). However, theadditional resolution obtained with two-photon tomography
allows one to appreciate the cellular morphology as well, including
neuronal processes (Figure 8) and astrocyte morphology (Figures
10 and 11).
The additional cellular resolution of serial two-photon tomography (as compared to OPT) can be exploited for quantification,such as cell counting in an extended volume. As evaluation of the
3D antibody staining in sections indicated that the penetration of
the GFAP antibody was not as complete as the DCX and nestin
antibody penetration, we compared the quantification of celldensity in the two cases. We generated 2D area density maps fromindividual slices of the serial two-photon tomography GFAP
volume images and from individual GFAP-stained histologicalsections (Figure 11). A zoomed in image of the corpus callosum
shows similar staining for the 3D immunostaining method incomparison to the 2D method (Figure 11E and F); however, there
is fainter and less abundant staining in the hippocampus anddentate gyrus for the 3D method in comparison to traditional
immunostaining (Figure 11G and H), matching the previous
staining results. The density maps produced from the 3D staining
method can be compared to those obtained with traditional
histology (Figure 11K and 11M vs 11L and 11N). In the anterior
portion of the brain, the density maps are very similar. In theposterior portion, the density maps show that the 3D staining, asapplied here, is not detecting as many cells in parts of the
hippocampus (particularly in the dentate gyrus), but shows anincreased staining in the cortex in comparison to the 2D stained
tissue (Figure 11M,N).
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Figure 8. 3D serial two-photon tomography imaging of DCX staining in the mouse brain. Serial two-photon tomography was used toimage a single hemisphere stained with DCX. Five individual slices are shown, with the slice location shown in the cartoon at lower right. For eachslice, the grayscale image shows autofluorescence for anatomical reference and the color overlay shows the DCX staining. Neuronal processes can be
distinguished (DCX in grayscale insets i, ii; arrowhead, inset i). The staining pattern is comparable to traditional 2D sections (insets with red color, iv
and v), although with lower contrast to background signal.
doi:10.1371/journal.pone.0072039.g008

3D double staining enables visualization of two different
cell types simultaneously in the adult mouse brain

reason, we used the 3D staining method for simultaneous staining
with two antibodies, a common practice with traditional immunohistochemistry.
We used the nestin and DCX antibodies demonstrated
previously and expanded the staining protocol by running thestaining procedure twice in series (i.e., adding a second primary

While the ability to visualize the distribution of a single cell
population in a 3D context will be useful in its own right in manystudies, it is advantageous to be able to investigate the spatial
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3D Immunostaining and Imaging in the Mouse Brain

antibody soak, wash, secondary antibody soak, and wash following
the original protocol with the first antibody, using the same times
and temperature). We then imaged the specimens with OPT tovisualize the resulting stain for each cell population and theautofluorescence background to provide anatomical context(Figure 12).

Discussion
The vast majority of mouse studies rely on histology to assess
cellular events in disease, development, or aging. In particular, theability to visualize neural progenitor and related cells in 3D allows
for their spatial distribution to be analyzed over a large volume
and the results to be placed in a larger anatomical context. We
have established a staining procedure that allows for the mappingof doublecortin-, nestin-, and GFAP-positive cells in the SVZ,
SGZ, RMS, olfactory bulbs and cortex.
The protocol that we developed shares some similarities withother reported protocols. In particular, the freeze/thaw cycle andmild digestion of the extracellular matrix have been reported
elsewhere [13,15,21]. Our protocol additionally used a lightfixation (1% PFA rather than 4% PFA) and performed incubations
at 37uC, rather than at either 4uC or room temperature. Boththese steps provided improved diffusion of antibodies into thetissue. Note that, while we performed these incubations in ahistology microwave, it may be possible to the carry out the
protocol in a temperature-controlled waterbath as well. Thismeans that the 3D staining protocol can be implemented without
any specialized equipment or reagents and that it uses only
commercially available antibodies.
Generally speaking, the protocol we applied generically to all
three of the antibodies described should be adapted for each new
antibody. This empirical adjustment is already common practicein traditional immunohistochemistry. The selection of the

Figure 9. 3D serial two-photon tomography imaging of nestin
staining in the mouse brain. Serial two-photon tomography wasused to image a single brain hemisphere stained with nestin. The slice
locations are shown in the cartoon. The staining pattern (grayscale
inset) is comparable to the 2D histology section (color inset).
doi:10.1371/journal.pone.0072039.g009

Figure 10. 3D serial two-photon tomography imaging of GFAP staining in the mouse brain. Serial two-photon tomography was used toimage a single hemisphere stained with GFAP. Optical slices through the brain show the staining pattern, which is widespread throughout the brain.
The slice location is shown in the cartoon. The grayscale inset shows the typical astrocyte morphology, which is comparable to the 2D histology
section (colour inset).
doi:10.1371/journal.pone.0072039.g010

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Figure 11. Comparison of serial two-photon tomography in GFAP-stained brains and traditional immunohistochemistry insections. Slices from a 3D serial two-photon tomography data set (A, C) are compared with traditional sections (B, D). Expanded images are shownfor comparable regions (E, G, I and F, H, J). Rostral sections (A, B and E, F) show comparable results. Staining in the hippocampus is lighter in the 3D
stained section (G) than in the traditional section (H). At the periphery, the 3D stain produces more intense GFAP signal with a higher background (I)than does the traditional section (J). Examples of lightly-stained cells are highlighted with arrowheads in (G) and (J). The arrow in (I) highlights an
example of the characteristic astrocyte morphology. Maps of cell density are shown in (K, M). Rostral cell densities (K, L) are similar between the two
methods. In the caudal regions (M, N), the density in the dentate gyrus (white arrowheads) is reduced in the 3D stain while it is appears increased insome regions of the cortex (asterisks). Cell density maps (K–N) are shown with black and white corresponding to ,30 cells/mm2 and .1200 cells/
mm2 respectively.
doi:10.1371/journal.pone.0072039.g011

opment of such an automated and comprehensive quantification
will permit studies that might otherwise be prohibitively laborious.
Clearing of samples is a critical component of imaging with
OPT, LSFM or related techniques. In this work, stained brains
were cleared with BABB for imaging with OPT. Other processes
and agents have been reported that are better able to preserve thefluorescence from transgenically-expressed proteins and may posesome advantages for antibody staining as well [12,25,26]. A new
method called CLARITY, in particular, was published by the
Deisseroth group while this manuscript was in preparation. The
authors demonstrate immunostaining in the clarified brain. The
diffusion of antibodies into the brain was undoubtedly enhancedby the removal of lipids in the CLARITY method. The simple
modifications for staining that we have presented here arecomplimentary; the extended incubation times for immunostaining even with CLARITY can be further reduced by appropriate
selection of the concentration of formaldehyde/hydrogel and by
adjustment of the incubation temperature as described here.
Similarly, the processing associated with CLARITY may improvethe diffusion that we have observed, which was not complete insome cases. Short and reliable immunostaining methods for intact
brains will extend the applicability of 3D optical imaging in futurestudies.
Although we demonstrated brain imaging with OPT [1,5] andserial two-photon tomography [4], we anticipate that this method
could readily be applied to other 3D optical imaging modalities

antibody dilution and the duration of each step will likely dependon the antibody, the abundance of the protein, and the location of
structures of interest. For example, we found that the concentration of DCX antibody needed to be higher to visualize the SGZ
and the olfactory bulb then it did if only the SVZ and RMS wereof interest. Similarly, GFAP penetration to posterior brain regions
was slow, which we attribute largely to the high concentration oftissue antigen for GFAP, particularly in the pial layer surrounding
the brain [22]. The concentration of antigen likely also explains
the difference in the appearance of some punctate voxels/pixels
between the GFAP and DCX/nestin images. Thus, the region ofinterest and the concentration of antigen may be an importantconsideration in selecting antibodies for 3D staining and in
adapting the proposed 3D staining method. Once the final
concentrations and antibodies have been selected, we have foundthe protocol to be very repeatable for subsequent specimens.
The combination of immunostaining with 3D optical image
acquisition provides new opportunities for quantification of cell
distributions across populations. Specifically, the acquisition of 3D
digital volumes enables population comparisons through use ofcomputer automated registration for alignment of multiple image
data sets [1,23,24]. Anatomical features (obtained, for instance,from autofluorescence) can provide assessment of morphologicalphenotypes, and then the antibody specific image can provide
quantification of underlying changes in cell populations. Devel-

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Figure 12. 3D OPT imaging of DCX and nestin stained mouse brain. 3D optical imaging with OPT was used to image a single hemisphere
stained with DCX (red) and nestin (blue) simultaneously. Optical slices through the tissue are shown to represent the 3D nature of the sample (A). The
slice orientation is shown on a cartoon. A 3D representation shows the distribution of DCX- and nestin-stained cells along the surface of the lateral
ventricle (B).
doi:10.1371/journal.pone.0072039.g012(such as LSFM [7–9] or other block-face imaging techniques[10,11]). There are, of course, advantages and disadvantages toeach imaging type. In our case, the OPT imaging procedure itself
is very fast and the sample can be recovered for further analysis if
desired. On the other had, serial two-photon tomography is able toachieve higher resolution, and is much less affected by the
autofluorescence background or by depth within the tissue (owing
to serial cutting). In either case, the 3D staining method is readily
applicable in any laboratory with access to appropriate 3D opticalimaging tools and will expand the potential impact of these tools inbiomedical research.

Author Contributions
Conceived and designed the experiments: JAG RMH BJN. Performed theexperiments: JAG. Analyzed the data: JAG JPL BJN. Contributed
reagents/materials/analysis tools: JPL RMH BJN. Wrote the paper: JAG
JPL RMH BJN.

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