|
ACETYLCHOLINESTERASE- with HRP (Mesulam,
1976)
Perfusion:
Saline rinse at 21 degrees C. (50 ml for rats, 200 ml for monkeys). Continue
with fixative (500 ml for rats, 2000 ml for monkeys) containing 1.25%
glutaraldehyde and 1% paraformalde- hyde in 0.1 M phosphate buffer pH
7.4 and a temperature or 21 d.C. Follow with an equal volume of 4 degrees
C. buffer with 10% sucrose Brains may be stored for 24 hours at 4 degrees
C. Section at 40 um and collect in phosphate buffer.
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, pH5) |
50 ml
|
| |
Promethazine hydrochloride(Phenergan) |
12.5 mg
|
| |
|
|
| 1. Wash in D-H2O
|
1 min |
| 2. Incubate at 40 degrees C. in solution
of equal volumes of Solutions A and B |
60 min |
| 3. Wash 6x in D-H2O |
3 min total |
| 4. 1.25% sodium sulfite |
1 min |
| 5. Wash 6x in D-H2O |
3 min total |
| 6. 1% silver nitrate (insert HRP processing
here) |
1 min |
| 7. 5% sodium thiosulfate |
5 min |
| 8. Wash 6x in.D-H2O |
3 min total |
| 9. Mount sect1.ons, air dry, dehydrate
and coverslip. |
|
Horseradish Peroxidase:
Prior to development of AChE (step 7,5% sodium thiosulfate), develop the
HRP reaction product by following the procedures described under HRP
-with AChE.
|