Biomedical Engineering Reference
In-Depth Information
but the basic hybridization procedures described in the following have been
based on riboprobe preparations described originally by Young
(15
,
16)
and
Whitfi eld et al.
(17)
.
3.1. Brain Tissue Preparation and Sectioning
Immediately dissect out the animal brain specimens that will be used for the
ISHH analysis (
see
Note 1
). Freeze the brains by immersion into dry ice-cooled
(-32°C to -40°C) isopentane for 30 s (
see
Note 2
). Store the brains at -30 to
-80°C (preferably with desiccant) in a sealed container if the brains will not
be immediately sectioned that day. For human studies, the brain specimens
should be cut into blocks of tissue approx 1.5 cm thick of the brain areas (e.g.,
striatum) of interest. Freeze the brain blocks by immersion into dry ice cooled
(-32°C to -40°C) isopentane for 1 min and store frozen (with dessicant) at
80°C until use.
In preparation for cutting the brain sections, allow the brain to acclimate to
the cyrostat temperature (-18 to -20°C) for at least 1 h. Mount the brain onto
the cryostat pedestal on dry ice with embedding matrix in the orientation at
which the brain will be sectioned (e.g., coronal). Cut the sections (12-20
m
thick;
see
Note 3
), mount onto superfrost microscope glass slide (
see
Note 4
),
dry for approx 1 min on a slide warmer plate (30°C), and place the slide into
a slide box maintained at least -18°C. For the rat brain, serial sections can be
taken at only one level of the nucleus accumbens (e.g., 1.7 posterior to Bregma
[18]
or throughout the rostrocaudal extent of the striatum; sections from four
to six different striatal levels can be placed on the same microscope slide).
Store the section-mounted glass slides at -30 to - 80°C with desiccant until
the ISHH experiment is performed (
see
Note 5
).
µ
3.2. Brain Section Pretreatment
All solutions (including the ethanol) should be made with autoclaved DEPC-
treated distilled water. All containers should also be treated with DEPC for all
the prehybridization steps. The prehybridization conditions should be carried
out at room temperature, preferably under a fume hood (
see
Notes 6
and
7
).
1. Place slides in a slide holder and bring to room temperature (tissue retention to
the slides during the subsequent hybridization procedures can be improved by
incubating the slides at 37°C for about 10 min).
2. Fix the tissue by immersing the slides in 4% paraformaldehyde for 5 min, and
rinse slides twice in 1X PBS.
3. Acetylation of the tissue: Rinse the slides in 0.1
M
TEA-0.9% saline, pH 8.0.
During this rinse make a fresh solution of 0.25% acetic anhydride in 0.1
M
TEA-0.9% saline, pH 8.0. Incubate the sections in this solution for 10 min and
rinse subsequently in 2X SSC.
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