Hi,
I'm trying NADPD-diaphorase stain which we hope will stain cholinergic neurons in rat brain stem.
Unfortunately, the protocol which has been used in the lab is somehow gone.
However, I know that they did it as described in Scherer-Singler, 1983.
So I wonder if it's still state-of-the-art.
I found two other protocols in some lab manual, which are slightly different from each other. They go like this:
PROTOCOL I
NADPH-solution (84 mg/ml in distilled water)
NBT-solution (1 mg in 20µl ETOH and 20 µl dimethylformamide)
0.1 M Phosphat-Buffer (ph. 5.3)
1. preincubate 30 min in 0.3 % Triton X-100
2. add to this solution:
· 10 µl NADPH-Lösung (final 1 mM)
· 10 µl NBT-Lösung (final 0.3 mM)
3. inkubieren 90 min
4. wash thouroughly with 0.1 M Phosphat-Buffer
5. mount
And an other one which goes like this:
NADPH (100 mg/ml): 10 mg NADPH-Tetrazoliumsalt in 100 µl H2O, fresh!
NBT: suspend 50 mg Nitro Blue Tetrazoliumchlorid in 431 µl Dimethylformamid, add 185 µl H2O to solve
TRIS (500 mM): 12,2 g TRIS in 200 ml H2O, adjust pH to 7,6
TRIS-washing-buffer (50 mM): dilute 1:10 with H2O
Triton-X-100-solution (10 %): 2 g Triton-X-100 in 20 ml H20
Preincubating-solution: 1 ml TRIS-(500mM), add 9 ml H2O, add 50 µl Triton-X-100-solution (10%), adjust pH with 80 µl 1 N NaOH to 8.2
Incubating-solutin: take 10 ml preincubating-solution, add 100 µl NADPH-solution and 25 µl NBT-solution (final conc. 0.2 mg/ml)
1. wash slices 10 min in Tris-buffer
2. preincubate slices 10 in preincubation-solution
3. incubate slices 3-5 h at 37°C in incubation-solution, control
4. wash slices 10 min with Tris-buffer
5. take them to PBS
6. wash 2x in ETOH
7. 2x Xylol
8. mount
Naturally, I wonder which one is better suited to my purpose. And I do not know whether these two protocols are as good as the one from Scherer-Singler (1983).
If you look at them closely, there are quite big differences in pH and in the solutions themselves...
Are there other protocols around, or is Scherer-Singler still state of the art?
Thx,
Dierk
