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serially in 10, 20, and 30% sucrose in Dulbecco's modifi ed phos-
phate buffered saline (DPBS) for 24 h. Brains can then be cryosec-
tioned (25
m) on the axial plane and stored in DPBS with 0.1%
sodium azide until used.
μ
To visualize immunoreactivity, free fl oating brain sections are
stained using a modifi ed ABC procedure (Vector Laboratories;
Burlingame, CA). Sections are treated with 10% hydrogen perox-
ide in DPBS for 15 min to quench endogenous peroxidases.
Following 3× rinses in DPBS for 5 min, sections are incubated in a
permeabilizing solution (1.8%
L
-lysine, 4% normal horse serum,
0.2% Triton X-100) for 30 min at room temperature. Sections are
transferred directly to primary antibody solution with 4% horse
serum (rabbit anti-protein of interest; 1:400; Abcam; Cambridge,
MA) and incubated overnight at room temperature. The following
day, sections are rinsed 3× in DPBS for 5 min and transferred to
the secondary antibody for 2 h (anti-rabbit IgG; 1:1,000;
Invitrogen). Following 3× rinses in DPBS for 5 min, sections are
incubated in Avidin d-HRP (1:1,000; Vector Laboratories) for 1 h
at room temperature; rinsed 3× in DPBS, and incubated with Nova
Red (Vector Laboratories) for 5 min. Following a 5 min rinse in
distilled water, sections can be mounted onto microscope slides,
air-dried overnight, dehydrated through a standard ethanol series,
and coverslipped. Demonstrated in Fig.
3
.
2.2. Immuno-
histochemistry
Confocal assessment of blood brain permeability can be achieved
utilizing lysine fi xable dextran (70,000 Da; AlexaFlour 488) and
fi brinogen (300,000 Da; AlexaFlour 568). A milligram of marker
can be dissolved in 0.5 ml of 0.9% saline and injected into the femo-
ral vein during or preceding the injury model. Rats are anesthetized
and transcardially perfused with 0.9% saline for 5 min then 4% para-
formaldehyde/0.1 M sodium cacodylate buffer (pH 7.2). Brains
are removed and postfi xed in 4% paraformaldehyde overnight.
Areas of interest are cut into 4 mm blocks, sliced into 60
2.3. Confocal
Microscopy for
Blood-Brain Barrier
Assessment
m sec-
tions, and assessed for fl uorescent reactivity using a confocal micro-
scope and analyzed. This technique is demonstrated in Fig.
4
.
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Albumin extravasation can be accessed by infusion with 2% Evans
blue (4 ml/kg) via the femoral artery. The Evan's blue is allowed to
circulate for 1 h and the rats perfused with cold PBS (pH 7.4) for
15 min via the left ventricle. The brains are excised; meninges and
ependymal organs removed, hemispheres excised, separated,
weighed, and homogenized in 500
2.4. Evans Blue
Albumin Extravasation
l of 50% trichloroacetic acid.
The tissue is incubated for 24 h at 37°C, and then centrifuged at
13,000 ×
g
for 10 min. The supernatants are diluted fourfold with
absolute ethanol; fl uorescence intensity is measured using a fl uo-
rometer at 620 nm excitation, 680 nm emission (Beckman DU
640). Calculations are based on external standard readings and
extravasated dye expressed as ng EB/mg brain tissue.
μ
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