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Fig. 4. Phenotypic analysis of BrdU-labeled cells. (A) Four weeks after the last
injection of BrdU, cells double-labeled for BrdU and NeuN could be detected.
This image confirms previous findings that numerous BrdU/NeuN-positive cells,
which are morphologically indistinguishable from the surrounding granule cells
can be found 4 weeks after BrdU injection. The inset highlights the
double-labeling by showing the immunoreaction for NeuN (arrow) in the
BrdU-labeled cell. BrdU/NeuN-positive cells can be found in the entire GCL,
not only in the SGZ, indicating early expression of a mature neuronal marker
and migration soon after division. NeuN, green; BrdU, red; astrocytic marker
S100ß, blue. Scale bar: 80 µm. (B) Eleven months after BrdU injection,
BrdU/NeuN-labeled cells could be found. They were morphologically the same as
they were 4 weeks after BrdU injection (compare with B). Generally, the
staining intensities are lower in the older animals. The whitish granules in
and between the cells are age pigment lipofuscin. NeuN, green; BrdU, red;
astrocytic marker S100ß, blue. Scale bar: 20 µm. (C) BrdU-labeled
cells can be detected as early as 1 day after the last (of 12) injection of
BrdU (see inset for display of NeuN-immunoreaction, arrow). NeuN, green; BrdU,
red; doublecortin (DCX, see below), blue. Scale bar: 25 µm. (D) Twenty-four
hours after a single injection of BrdU, no BrdU/NeuN-labeled cells can be
detected (arrow in upper inset; compare with top left inset). Furthermore,
NeuN cannot be detected in cells that express intermediate filament nestin as
a marker of stem or progenitor cells. Nestin was detected in these mice by
expressing green fluorescent protein (GFP) under the nestin promoter
(Yamaguchi et al., 2000 ). The
nestin-GFP-expressing putative stem or progenitor cells form clusters along
the SGZ (see lower inset). GFP, green; BrdU, red; NeuN, blue. Scale bar: 15
µm. (E) DCX is a protein expressed in young, maturing neurons. Soon after
division, many BrdU-labeled nestin-GFP expressing cells become DCX positive.
The lower panel shows the fluorescence intensities along the horizontal line
drawn through one of the nuclei. BrdU, red; nestin-GFP, green; DCX, blue. The
marked cell is triple-labeled, but the fluorescence intensity is lower than in
the right neighboring cell that expresses nestin-GFP only. Scale bar: 25
µm. (F) Colocalization of BrdU and DCX can be further visualized in
z-series through the section (z-distance is 12 µm). The
reconstructed views along the yz-axis (right panel) and
xz-axis (bottom panel) demonstrate that red BrdU immunoreaction is
surrounded by blue DCX immunoreaction. NeuN, green. Scale bar: 20 µm. (G)
The overview shows that along the SGZ BrdU-positive cells (red) can be found
that express DCX (blue) and appear pink (arrow, see also F). Similar to C,
some BrdU/NeuN-positive cells (orange, arrowhead) can be seen. DCX-labeled
cells are restricted to the SGZ. NeuN, green; BrdU, red; DCX, blue. Scale bar:
80 µm. (H) Many DCX-expressing cells are NeuN-positive, indicating a
temporal overlap between the two markers. DCX-expressing cells often have long
processes, extending into the molecular layer. The inset indicates how
cytoplasmic DCX expression (blue) engulfs nuclear and perinuclear
NeuN-expression (green). BrdU, red. Scale bar: 10 µm. (I)
ß-III-tubulin can serve as another marker for immature neurons. At early
time-points after BrdU, clusters of BrdU/ß-III-tubulin marked cells can
be found. However, ß-III-tubulin yields a weaker staining, does not
extend into the neurites and cannot be combined with NeuN. ß-III-tubulin,
green; BrdU, red; S100ß, blue. Scale bar: 20 µm. (K) New astrocytes
can be identified by colocalizing immunoreactivity for BrdU (red) and
S100ß (blue). NeuN, green. Scale bar: 15 µm. Insets in I and K show
controls.
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