Immunohistochemistry
For the human SVZ, we performed immunohistochemistry on 8 μm-thick frontal sections of
paraffin-embedded striatal material as described before (van den Berge et al., 2010). In brief,
sections were rinsed with Tris-buffered saline (TBS) and steamed in citrate buffer (GFAP-δ, pHH3 and
tyrosine hydroxylase (TH); 10mM citric acid + 0.05% Tween 20, pH 6.0, 98°C) or Tris/EDTA buffer
(PCNA, 10mM Tris Base, 1mM EDTA, 0.05% Tween 20, pH 9.0, 98°C). For staining of GFAP-δ, pHH3
and PCNA, sections were pre-incubated with horse serum-containing buffer, and incubated overnight
in the primary antibody solution (see supplemental table 1). After peroxidase blocking, sections were
subsequently incubated with a biotinylated secondary antibody, avidin-biotin complex (ABC) and
diaminobenzidine tetrahydrochloride (DAB). For the TH staining, steamed paraffin sections were
incubated in 0.3% H2O2 in 50% methanol for 10 min and washed with TBS. Subsequently, the sections
were pre-incubated for 30 min with 5% non-fat dry milk in TBS containing 0.5% Triton-X100 (TBS-T).
Thereafter, sections were incubated overnight at room temperature with the primary antibody (see
supplemental table 1). After 24 hours, the antibody solution was refreshed and sections were again
incubated overnight. After washes in TBS, the sections were incubated for 60 minutes at room
temperature with horse anti-mouse biotin (1:100, Jackson, Suffolk, UK). Following washes in TBS,
sections were incubated for another 60 min at RT with ABC-HRP complex. Sections were washed
twice with TBS, once with Tris-HCl and peroxidase activity was visualized by DAB and sections were
mounted with entellan. For TH staining, all antibodies were diluted in TBS containing 0.5% TritonX100 and 5% non-fat dry milk.
For the olfactory bulb, 20 µm-thick sections of paraffin-embedded material were stained for
GFAP-δ as described before (van den Berge et al., 2010). In brief, sections were steamed in TBS
(98°C), blocked for endogenous peroxidase, and pre-incubated with horse serum-containing buffer.
Then, they were incubated with GFAP-δ antiserum, biotinylated secondary antibody, ABC, and DAB.
For the mouse SVZ stainings, 30 μm-thick coronal vibratome sections of the whole brain
were cut and stored in 1% PFA. Before the start of the free-floating staining procedure, the sections
were washed in TBS with 0.2% Triton X-100 (TBS-Tx) for 10 minutes. Then, they were incubated in
sodium citrate buffer (50mM tri-sodium citrate, 0.05% Tween 20, pH 6.0, 98°C) in a steamer for 20
minutes. After cooling down, the sections were washed again in TBS-Tx and pre-incubated with block
mix (TBS-Tx containing 2.5% donkey serum (DAKO A/S, Glostrup, Denmark)) for 1 hour. The sections
were incubated with primary antibodies (see table 1) diluted in block mix for 1 hour at room
temperature and for 16-24 hours at 4°C. After washing with TBS-Tx, sections were incubated with
fluorescently labelled secondary antibodies (Jackson Immuno-Research Laboratories, Inc., West
Grove, PA, USA; 1:400) and Hoechst 33258 (BioRad, Hercules, CA, USA; 1:10000) in block mix. Finally,
the sections were washed in TBS, mounted on chromic potassium sulfate-coated glass slides, and
embedded in Mowiol (0.1 M Tris pH 8.5, 25% glycerol, 10% w/v Mowiol 4-88 (Sigma)).
Fixed CB660 cells were washed with TBS and steamed in citrate buffer before incubation with
BrdU antibody (see table 1). After 16 hours, the cells were washed, and incubated with the
secondary antibody coupled to Alexa546 and Hoechst, before embedding them in Mowiol.
Image acquisition and quantification
Images of single immunostainings were obtained on an AxioSkop microscope (Zeiss,
Oberkochen, Germany) with Neoplanfluor objectives, using a Sony XC77 black and white camera
(Sony, San Diego, California) and ImagePro software (MediaCybernetics, Bethesda, USA). Images of
immunofluorescent staining were obtained on an AxioPlan 2 microscope (Zeiss) with
Planapochromat objectives, using an Evolution QEi black and white camera (MediaCybernetics) and
ImagePro software.
The number of dopaminergic neurons in the substantia nigra was estimated using designbased stereology. We determined the number of neuromelanin-containing neurons in Nissl-stained
20 m thick paraffin sections or 40 m thick frozen sections collected throughout the
mesencephalon of 4-5 cases per group. Neuromelanin-containing neurons were counted in random
and systematically sampled sections throughout the left or right substantia nigra. The substantia
nigra was delineated at low magnification (25x UplanApo objective) using the coordinates of the atlas
of the Human Brainstem (1st edition, 1995) using a computer-assisted morphometry system
consisting of a Zeiss Axioplan photomicroscope with a CCD color video camera and a motorized stage
controller for automatic sampling equipped with the StereoInvestigator software version 8
(MicroBrightfield Inc., Colchester, VT). A LEP XY motorized stage controller controlled the movements
along the x- and y-axes, and a Heidenhain MT12 microcator attached to the stage measured the
precise focal depth, with a resolution of ~0.5 μm. Estimates of the total number of neuromelanincontaining neurons were made using the optical fractionator method (West et al., 1991) with a 40x
objective. To prevent experimenter bias, all sections were coded. Neuromelanin-containing neurons
were counted if the nucleus came into focus within the counting frame. Analysis was performed
starting with the first anterior appearance of neuromelanin-containing neurons (obex +47 mm) and
extending to the most caudal parts of the substantia nigra (obex +34 mm). The following sampling
parameters were used: ssf = 1/10 for frozen tissue and 1/12 for paraffin-embedded tissue, the height
of the dissector = 10 μm, the guard height = 2μm, the counting frame area = 10,000 μm2 and asf =
0.04, as defined by the counting frame area multiplied by the reciprocal of the grid size (400 x 400
μm). The average predicted coefficient of error of the estimated total number of neurons CEpred was
0.058 (Schmitz and Hof, 2005). The mean estimated thickness of the paraffin and frozen sections
after histological preparations was respectively 17.2 ± 0.97 and 13.8 ± 2.1. The determine the density
of neuromelanin-containing neurons within the substantia nigra, we divided the total estimated
number of neuromelanin-containing neurons by the total measured volume per donor.
For quantification of PCNA, GFAP-δ, and TH expression in the human SVZ, 5 images were
taken at random locations of the SVZ. In these images, an area of interest was drawn around the area
of staining, excluding the ependymal layer, and the background intensity was measured. Then, a
threshold, at which specific staining was identified, was set, to calculate the surface area of protein
expression. The length of the subventricular zone in the image was also measured. For the
quantification, we calculated the average percentage of SVZ area that was occupied by PCNA staining
in the 5 images. pHH3 cell numbers were counted manually in 5 randomly taken images of the SVZ,
averaged, and corrected for the length of SVZ that was studied.
For quantification of GFAP-δ in the human olfactory bulb, the entire bulb was imaged and
outlined. Then, a threshold of 5 times the background intensity was set to calculate the surface area
of GFAP-δ expression. This was done at various levels of the olfactory bulb, and these values were
averaged per donor.
For quantification of PCNA and tyrosine hydroxylase (TH) staining in the mouse SVZ, the
striatal SVZ was photographed throughout the striatum in coronal sections. In these images, an area
of interest was drawn around the area of PCNA staining and the background intensity was measured.
Then, a threshold of 1.5 times the average background was set to calculate the surface area of PCNA
expression, which was averaged over several sections. In the same area of interest, the intensity of
the TH staining was measured. pHH3 cell numbers were counted manually throughout the SVZ, and
corrected for the length of the SVZ.
For quantification of neurosphere proliferation, ihNSC cultures were photographed using an
Axiovert 200M microscope (Zeiss) with Planapochromat objectives, using an EXi Aqua camera
(QImaging, Surrey, BC, Canada) and ImagePro software. Per well, 5 images were made in a cross
pattern. The diameter of all neurospheres in these images was measured, as well as the number of
neurospheres in these images, and averaged per well. We repeated this experiment three times and
then normalised the data by setting the condition with growth factors and without agonists to 100%.
Proliferation of CB660 cells was measured by capturing 5 images per coverslip. These images were
analysed using ImagePro software, which automatically detected the BrdU-positive cells and
Hoechst-positive cells. These numbers were divided to obtain a BrdU-labelling index per coverslip.
This experiment was repeated twice and normalised by setting the condition with growth factors and
without dopamine to 100%.
For statistical analysis, the program SPSS Inc 17.0 was used. For the data from the human
SVZ, substantia nigra and olfactory bulb, all tests performed were non-parametric analyses, as the
data were not normally distributed (as determined using the Kolmogorov-Smirnov and Shapiro-Wilk
tests). Data from the mouse SVZ were normally distributed, so parametric tests were performed.
Supplemental table 1. Antibodies used.
Antibody
Species
Manufacturer
Dilution
β-III-tubulin
mouse monoclonal
Sigma SDL.3D10
1:400
BrdU
mouse monoclonal
DAKO M0744
1:1500
Galactocerebroside
rabbit polyclonal
Chemicon AB142
1:200
GFAP- δ
rabbit polyclonal
NIN 100501 (Roelofs et al., 2005)
hSVZ 1:500, hOB 1:100
pan-GFAP
rabbit polyclonal
DAKO Z0334
1:1000
PCNA
mouse monoclonal
Santa-Cruz PC10 sc-56
SVZ 1:2000, hOB 1:100,
pHH3
rabbit polyclonal
Sigma H 0412
hSVZ 1:800, mSVZ 1:1000
Tyrosine Hydroxylase
rabbit polyclonal
PelFreez P40101-0
mSVZ 1:1000
Tyrosine Hydroxylase
mouse monoclonal
Chemicon MAB318
hSVZ 1:50
hSVZ = human subventricular zone, hOB = human olfactory bulb, mSVZ = mouse SVZ
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