Biology Reference
In-Depth Information
Thin, sharp blades (Leica disposable microtome blades, Cat. 63065).
2.4. Staining Equipment
and Solutions
Hematoxylin and eosin solution (Sigma Aldrich Co., St. Louis, MO).
Distilled water.
Staining racks.
Alcohol solutions (50-100%) for dehydrating tissue.
2.5. Photography
and Processing Tools
Light microscope and camera for image acquisition or a micro-
scope slide scanner.
Calibrating scale.
Software for image analysis, such as ImageJ—National Institutes of
Health (NIH) imaging software (NIH, Bethesda, MD).
3. Procedures
Prepare 4% paraformaldehyde and saline solutions (0.9% NaCl
solution).
Perfuse rodent according to standard protocols; use saline to fl ush
out blood, followed by 4% paraformaldehyde for fi xation.
After 20 min of controlled and pressurized perfusion, extract the
brain carefully and postfi x in 4% paraformaldehyde for 2-3 days
before embedding in paraffi n.
3.1. Fixation and Brain
Extraction
Remove the rodent brain carefully from the skull, carefully excise
the dura matter and place onto a Petri dish.
Place the brain onto the matrix, dorsal side up.
Place one single-edged razor at the cerebro-cerebellar junc-
tion, followed by the placement of a second razor 2 mm cau-
dal for a brain stem and basilar artery (BA) section.
Turn the frontal part of the brain ventrally to expose the circle of
Willis.
Two longitudinal cuts are performed across the middle cerebral
artery (MCA), followed by one transverse cut for the anterior
cerebral artery (ACA, Fig.
1
).
Sections are put in cassettes and embedded in paraffi n using stan-
dard methods.
Sections 7-10
3.2. Brain Sectioning
μ
m thick are cut on a microtome.
3.3. Hematoxylin
and Eosin Staining
Place slides with sections on them into a staining rack.
Sections are deparaffi nized in xylene and rehydrated through a
decreasing gradient of ethanol solutions (100-70%).
Slides are stained with hematoxylin and eosin and coverslipped
with xylene-based mounting medium.
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