Biomedical Engineering Reference
In-Depth Information
Fig. 3
Dynamics of HSV-1 migration in the brain after injection into the DRt. Time-course analysis of
ʲ
-gal
expression into the brain after DPZ injection into the DRt (
a
). Photomicrographs of
-gal-positive neurons in
the cerebellum (
b
) and the locus ceruleus (
c
) at 7 days p.i. Scale bar in
b
: 100 µm (
a
is at the same
magnifi cation)
ʲ
and 14 days postinjection (p.i.) (Fig.
3a-c
) [
6
]. The immunodetection
of the
ʲ
-gal expressed by DPZ is performed as follows:
1. After incubation in 0.1 M PBS containing 0.3 % Triton X-100
(PBS-T) containing 0.1 M glycine and 10 % normal swine
serum for 2 h to decrease background staining, incubate the
sections overnight with a monoclonal anti-
-gal antibody
raised in mouse (Roche, USA) diluted at 1:5,000 in PBS-T
containing 2 % normal swine serum.
2. Wash with PBS-T, and then incubate for 1 h with a rabbit bio-
tinylated anti-mouse serum at 1:200 (Dako, Denmark) diluted
in PBS-T containing 2 % normal swine serum.
3. Wash sections again with PBS-T and incubate for 1 h in PBS-T
containing the avidin-biotin complex (1:200; ABC; Vector
Laboratories, USA).
4. Wash in 0.1 M Tris-HCl, pH 8.2; then to reveal bound peroxi-
dase, incubate with 0.0125 % 3,3
ʲ
-diaminobenzidine tetrahy-
drochloride (DAB; Sigma Immunochemicals, USA) and
0.025 % H
2
O
2
in the 0.1 M Tris-HCl, pH 8.2 buffer.
5. Mount sections on gelatin-coated slides, dehydrate in xylene
for 5 min, and coverslip with Eukitt (Sigma, Sigma Immuno-
chemicals, USA).
′
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