Biology Reference
In-Depth Information
3.2. Brain Sectioning
For rats,
Section the brain at 18-
m thickness by a cryostat.
Label brain sections and keep brain sections in −80°C freezer.
μ
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For pigs,
Cut the brain by sharp blade.
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Place the sections on a piece of blue-pad, line up the slices
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from anterior to posterior, with the left hemisphere on the left
with a ruler.
Take photos with the digital camera (do not use fl ash; use
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macro close-up mode).
Embed brain slices in paraffi n after the sections are dehydrated
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in the tissue processor (Leica ASP300) under the automatic
programs.
Section the blocks at 7-
m thick on Polycut.
Dry the brain sections in the warm incubator at 60°C for at
μ
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least 24 h and store at room temperature.
3.3. Staining
Dry the brain slides at room temperature for 30 min.
Put the slides into hematoxylin solution for 30-50 s.
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3.3.1. H&E Staining
Rinse the slides in deionized water for 2 min for four times.
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Put the slides into eosin solution for 2 min or longer.
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Rinse the slides in deionized water for 2 min for four times.
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Dehydrate with ethanol at graded concentrations of 75-100%
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1 min.
Clear slides with 50% xylene in ethanol and 100% xylene for
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2 min.
Mount the slides with coverslip slides by Permount (Fisher
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Scientifi c, USA).
Dry the slides overnight in the hood.
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Deparaffi n the slides in 100% xylene for 15 min, 100% alcohol
for 10 min, 95% alcohol for 5 min, 70% alcohol for 5 min, and
distilled water for 5 min.
Unmask the slides in 0.01 M pH 6.4 citric solution and boil in
3.3.2. Luxol Fast
Blue Staining
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microwave over about 1 min.
Cool down the slides at room temperature.
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Repeat steps as in deparaffi nization.
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Dip the slides into Luxol fast blue solution overnight at
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60°C.
Rinse off excess stain with 95% alcohol.
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Rinse in distilled water.
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