The NADPH-diaphorase activity is the generic name for any enzyme activity able to transfer electrons between NADPH or NADH and several types of acceptors, such as the tetrazolium salts. Reduced tetrazolium salts become insoluble and precipitate as formazans with a blue color. This procedure labels those cell structures containing any dehydorgenase that oxidizes NADPH and reduces tetrazolium salts. For example, it has been used to uncover succinate dehydrogenase enzymes.
In the nervous system, NADPH-diaphorase activity is present in many neuronal populations that express the neuronal nitric oxide synthase. These neurons release nitric oxide as a uncoventional neurotransmitter. The NADPH-diaphorase activity also labels the endothelial nitric oxide synthase found in the blood capillaries. By in situ hybridization, it has been shown that every NADPH diaphorase positive neuron expresses the nitric oxide synthase enzyme. Thus, it is possible to study the neurons expressing this enzyme with a simple and easy procedure. However, the results have to be interpreted with caution because the labeling depends on the fixative and fixation process.
Procedure
Samples are fixed with buffered 4 % paraformaldehyde. 50 µm thick sections are obtained with a vibratome or freezing microtome.
1. 2x10 min in phosphate buffer 0.1 M pH 7.4 (PB).
2. 2x10 min in PB containing 0.3 % Triton X-100. Gentel agitation.
3. 10 min in PB + 0.3 % de Triton X-100 + NADPH (0.5 mg/ml) + Nitroblue tetrazolium (0.2 mg/ml).
Gentel agitation
Room temperature
In darkness
4. 2-6 h at 37 ºC in the solution of step 3.
5. Check periodically and stop with washes in PB
6. Dehydration and mounting.
Free-floating sections can be mounted by attached to gelatin coated slides. Use small brushes to handle the sections.
Results
Positive neurons, dendrites, axons: blue.
Postive blood vessels: blue.
Notes
Sections have to be thoroughly rinsed after the reaction. Otherwise the backgound increases with time.
If the developing process last long, formazan precipitates may appear. This can be prevented by periodically replacing the developing solution with a fresh one.
Products
Xylene
50º, 70º, 80º, 90º, 96º and 100º ethanol
Nitroblue tetrazolium salts
NADPH
Buffer phosphate 0.1 M pH 7.4
Triton X-100
Distilled H2O
Mounting medium
Labware
Staining dishes
Absorbent paper
Small brushes
Aluminium paper
Agitator
37 ºC stove
Gelatin coated slides
Coverslips
