Biology Reference
In-Depth Information
3.4. Brain Sectioning
1. Cut serial coronal brain sections (8 or 20
μ
m) thick using
cryostat.
2. Thaw-mount onto microscope slide (Colorfrost Plus; Fisher,
USA) one section every 1 mm starting; bregma at +2.7 to −6.7.
3. Fix sections on to the slide with chilled PAF 4% for 5 min.
4. Wash with PBS for 5 min.
5. Mount using aqueous mounting medium (Vector, Burlingame,
CA, USA) and cover slip.
1. Examine FITC-dextran labeling in sections via fl uorescence
microscope.
2. Take continual images of the whole coronal sections using a
fl uorescence microscope equipped with automated stage and
camera.
3. Alternatively, separate images (at least three per area) of stria-
tum, basal and frontal cortex per right and left hemisphere can
be taken.
3.5. Imaging
3.6. Image Analysis
1. Whole coronal sections: a montage of images showing the
whole coronal sections is created using MetaMorph (Molecular
Devices) and used for analyses (Fig.
1a
).
2. Alternatively, individual images can be analyzed (Fig.
1b
).
3. For analysis, in IPLab software (MetaMorph can be used for
this purpose as well).
1. Select whole image as ROI.
2. Segment the image by intensity to select tracer-labeled vessels.
3. Determine the number, total area, and area fraction of select
vessels.
4. Export the data to excel for statistical analysis.
3.7. Presentation
and Statistical
Analysis
1. Defi cits are presented as the number or area fraction of the
tracer-labeled vessels (
17
).
2. Comparison is made between the results from SAH and the
time matched sham-operated animals.
3. Data is analyzed via two-way ANOVA followed by a pair wise
comparison by a suitable (such as Fisher's PLSD) post-hoc test.
4. Cerebral
Microdialysis
4.1. Introduction
Cerebral microdialysis is a sampling technique that measures extra-
cellular concentration of substances by means of a small probe
equipped with a semipermeable membrane inserted into the brain.
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