Biomedical Engineering Reference
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Fig. 3. Histological examination of NAc after gene transfer.
(A)
Expression of
CREB at the completion of a place conditioning study, 5 d after microinjection of
HSV-CREB into the left NAc shell (
×
40).
Arrow
indicates injection site; scale bar
= 1 mm.
(B)
Higher magnifi cation (
200) of the area indicated by the box in
(A)
,
confi rming nuclear localization of CREB expression.
(C)
An adjacent, Nissl-stained
slice from the same brain, showing minimal evidence of toxicity or damage despite
strong transgene expression.
(D)
Expression of mCREB 5 d after microinjection of
HSV-mCREB into the NAc. Expression of mCREB, which contains a point mutation
at Ser133 that prevents phosphorylation, is indistinguishable from that of CREB with
the antibodies used. AC, anterior commissure.
×
3. Fix the rat with ice-cold 4% paraformaldehyde (for detection of
-galactosidase,
2% paraformaldehyde works best) using a low fl ow rate. Generally, fi xation
requires 300-500 mL of paraformaldehyde.
4. Postfi x the brain for 1-2 h in paraformadehyde solution.
5. Cryoprotect the brain overnight in 20% glycerol-50 m
M
PBS. When fi rst placing
the brain in this solution, it should fl oat. Do not section the brain unless it has
sunk into the solution (usually within 12 h). For best results, section the brain
within 48 h of fi xation.
3.5.2. Harvest Brain Tissue for Immunoblot
1. Remove the brain as rapidly as possible, and momentarily cool the whole brain
by placing it into ice-cold aCSF buffer.
2. Use a tissue slicer to obtain a 1-mm thick section of brain that includes the NAc
(i.e., 1.0-2.0 mm anterior to bregma;
see
ref.
5
).
3. Place the 1-mm thick section into a shallow Petri dish fi lled with ice-cold aCSF.
Use a 14-gauge tissue punch to obtain the NAc.
4. Place the tissue punches in prelabeled microcentifuge tubes. Flash freeze the
tissue by placing the tubes in ground dry ice.
5. Perform Western immunoblotting analyses as described in
Subheading 3.3.
).
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