笔记详情
标题
Oxidative modification of peroxiredoxin is associated with drug-induced apoptotic signaling in experimental models of Parkinson disease.
内容
Tissue Processing, Immunohistochemistry, and Quantitation—Animals were perfused transcardially with a saline solution containing 0.5% sodium nitrate and heparin (1000 units/ml) prior
to fixation with 4% paraformaldehyde dissolved in 0.1 m
phosphate buffer. Brains were removed and postfixed overnight in
buffered 4% paraformaldehyde at 4 °C, incubated in a 30%
sucrose solution for 48-72 h at 4 °C until they
sank, and sectioned frozen on a sliding microtome in 30-μm-thick coronal
sections.
Sections were collected and processed for
immunohistochemical staining for tyrosine hydroxylase (TH) alone or in
combination
with the oxidized forms of PRX. The unbiased
stereological estimation of the total number of TH-positive cells in the
substantia
nigra was carried out using the optical
fractionator, as previously described in full detail (29).
The sampling technique was not affected by tissue volume changes and
did not require reference volume determinations. Sampling
was done using the Computer-Assisted Stereological
Toolbox system, version 2.1.4 (Olympus Denmark A/S, Ballerup, Denmark),
using a microscope with a motorized stage run by an
IBM-compatible computer. The substantia nigra was delineated at a ×1.25
objective and generated counting areas of 150 × 150
μm. A counting frame (1612 μm2) was placed randomly on the
first counting area and moved through all counting areas until the
entire delineated area was
sampled. Actual counting was carried out using ×100
oil objective. Guard volumes (4 μm from the top and 4-6 μm from the
bottom
of the section) were excluded from both surfaces to
avoid the problem of lost caps, and only the profiles that came into
focus
within the counting volume (with a depth of 10 μm)
were counted. For double immunofluorescent localization of TH and the
oxidized
forms of PRX, tissue sections were further fixed in
100% methanol for 20 min at -20 °C, permeabilized in 0.2% Triton X-100
in 0.2
m phosphate buffer at room temperature for 1 h, blocked in 3% bovine serum albumin in 0.2 m
phosphate buffer at room temperature for 15 min, and then incubated
overnight at 4 °C with mouse monoclonal anti-TH (1:2000)
and rabbit polyclonal antibody against the oxidized
form of PRX (1:200). Tissue sections were rinsed extensively with 0.2
m phosphate buffer and incubated at room temperature for 1 h with a mixture of Alexa Fluor 568-conjugated goat anti-mouse IgG
and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200; Molecular Probes). Following extensive wash with 0.2 m
phosphate buffer, stained tissue sections were examined with an
AxioImager D1 fluorescence microscope equipped with a digital
image analyzer (Carl Zeiss) or an LSM 510 Meta
Laser Scanning Microscope (Carl Zeiss). To assess the number of
TH-positive
DA neurons or TH-negative cells co-localized with
the increased level of the oxidized forms of PRX, manual counts were
conducted
using 10-15 randomly selected confocal images per
condition by an individual who was blind to the treatment conditions.
Adjacent
tissue sections from each animal were also stained
with cresyl violet and subjected to quantitation to validate
immunohistochemical
determination of nigral neuronal survival.
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来源
J Biol Chem. 2008 Apr 11;283(15):9986-98.
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