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ACETYLCHOLINESTERASE -with HRP (Hardy
et al., 1976)
Perfusion:
flush briefly with 0.05 M phosphate buffer pH 7.4, follo~ with 4% glutaraldehyde
in 0.05 M phosphate buffer pH 7.4. Both solutions should be ice cold.
Section brains as early as 2 h. after perfusion. Cut sections at 50 urn;
collect in ice-cold phosphate buffer saturated with sodium sulfite.
Acetylcholinesterase:
| Solution A: |
0.226 g Acetylthiocholineiodide in 100
ml D-H2O |
|
| Solution B: |
|
|
| |
Stock glycine (80 mM) |
25 ml
|
| |
Stock CuSO4 (16 mM) |
25 ml
|
| |
0.2 M Acetate buffer (30 ml acetic acid,
70 ml Na acetate, pH 5) |
50 ml
|
| |
Promethazine hydrochloride (Phenergan)
|
12.5 mg
|
| |
|
|
| 1. Incubate 30 min. at 40 degrees C.
in solution of equal volumes of Solutions A and B. |
|
| 2. Wash 6 times in D-H20 (30 sec. each) |
3 min |
| 3. 1.25% sodium sulfite |
1 min |
| 4. Wash 6 times in D-H20 (30 sec. each) |
3 min |
| 5. 1% silver nitrate |
1 min |
| 6. Wash 2 times in D-H20 (30 sec. each) |
1 min |
| 7. 5% sodium thiosulfate |
5 min |
| 8. Wash 6 times in D-H20 (30 sec. each) |
3 min |
| |
|
Horseradish Peroxidase:
1. Incubate 5 min in a solution of 5% sucrose and 0.03% DAB in 0.05 M
Tris buffer.
2. Add 5 ml of 0.3% hydrogen peroxide to each 50 ml of incubation solution.
Shake 5-20 min or until pink.
3. Wash 2x in 0.05 M phosphate buffer, lx in D-H20.
4. Collect in 10% formalin, mount on glass slides and counterstain with
cresyl violet.
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