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.
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).
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.
Positive neurons, dendrites, axons: blue.
Postive blood vessels: blue.
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.
50º, 70º, 80º, 90º, 96º and 100º ethanol
Nitroblue tetrazolium salts
Buffer phosphate 0.1 M pH 7.4
37 ºC stove
Gelatin coated slides