Biomedical Engineering Reference
In-Depth Information
3. On the next day, continue the incubation with primary antibody at room tem-
perature for 0.5-1 h. Rinse four times in 0.02% Triton X-100-1X PBS (10 min
each) to wash off free primary antibodies.
4. Dilute the secondary antibody (biotinylated goat anti-rabbit from the Vector
ABC kit) 1
200 in the secondary antibody dilution buffer (0.02% Triton
X-100-1X PBS).
5. Incubate in the diluted second antibody solution for 1 h. Rinse three times in 1X
PBS (10 min each) to wash off free secondary antibodies.
3.4. ABC Complex, Staining and Mounting
1. Mix one drop each of reagent A (avidin) and B (biotinylated horseradish
peroxidase) (from the Vector ABC kit) in 10 mL of 0.02% Triton X-100-1X PBS
for 15-30 min. This allows the formation of the avidin-biotinylated horseradish
peroxidase complex (
see
Note 7
).
2. Incubate in the mixture for 1 h and rinse three times in 1X PBS (10 min each).
3. Incubate in the freshly prepared staining solution (0.25 mg/mL of DAB, 0.01%
H
2
O
2
, and 0.04% NiCl
2
) for 5-6 min. Closely monitor the intensity of the
reaction to ensure that background immunostaining remains at a minimal level
(
see
Note 8
).
4. Once the desired level of staining has been visualized, stop the reaction in
1X PBS. Rinse the section three times in 1X PBS (10 min each).
5. Remove fl oating sections to a Netwell mounting tray half fi lled with 0.1
M
sodium nitrate using a fi ne-tipped paint brush. Mount sections onto the gelatin-
coated glass slide. Several drops of 10% Triton X-100 can be added to facilitate
the moving of tissue onto the slide. For culture cells, remove mounted chambers
from glass slides carefully.
6. Slides are air-dried at room temperature to allow the adhesion of sections to
the surface of the glass slide. The DAB reaction product is essentially insoluble
and very stable. Immerse slides in distilled H
2
O for 1 min to remove the salts
remaining from the buffers previously used. If necessary, a counterstaining can
be performed here (
see
Note 9
). Dehydrate sections in graded ethanol (70%,
80%, 95%, and 100%; 1 min each). Ethanol is generally used to remove the
water in the tissue to allow the mounting media that is not miscible with water
to coverslip slides.
7. Place the slide in xylene to prevent the tissue from drying out and coverslip
with the DPX mounting media. Let slides dry overnight before examination
under a light microscope.
3.5. Anticipated Results in the Striatum
Using the procedures outlined in the preceding, the following characteristics
can be anticipated (for details,
see
refs.
5-8
). It should be mentioned that
experimental manipulations that can enhance striatal cellular activity need
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