Neuromuscular CLINICAL LABORATORY

NADH-TR "diaphorase" PROTOCOL

PRINCIPLE:

"Diaphorase" is a term given to flavoprotein enzymes that have the property of transferring hydrogen from reduced nicotinamide adenine dinucleotide (NADH) to various dyes. The hydrogen transfer reduces the dye. Usually tetrazolium compounds function as the hydrogen acceptor when diaphorases are being demonstrated histochemically, and the product of the reduction is the water-insoluble formazan pigment. Commonly used tetrazoliums include nitro blue tetrazolium (NBT). Enzymatic activity releases hydrogen from the substrate, and the released hydrogen is transferred to the tetrazolium. With the addition of hydrogen, the tetrazolium is converted to purple-blue formazan pigment marking the site of enzyme activity.

SPECIMEN REQUIRED:

Snap frozen human striated muscle. (Use the isopentane freezing method previously described.)

Controls: Use skeletal muscle or myocardium for a positive control. For a negative control, eliminate the substrate from the incubating medium.

METHOD:
Fixation: None, use snap frozen tissue.
Technique: Cut 10 - 16 micron (12 m) sections in cryostat from snap frozen biopsy. Attach one or more sections to a No. 1, 22 mm square coverslip.

Equipment:
Ceramic staining rack - Thomas Scientific #8542-E40
Columbia staining dish - Thomas Scientific #8542-C12
Columbia staining dish(jar) - Thomas Scientific #8542-E30
Forceps
Latex gloves

Reagents:
Acetone - Baxter #010-4, FLAMMABLE
Deionized water
Gelatin - 100 bloom -ICN 960317 store at room temperature
Glycerol -Sigma G 8773, store at room temperature IRRITANT
Nicotinamide adenine dinucleotide, reduced (NADH)- Sigma N8129
Nitro blue tetrazolium - Sigma N6876), store at 0- 5 o C Phenol - Fisher A931-1, CAUSTIC, Store at room temperature
TRIS base (Tris[hydroxymethyl]aminomethane) - Sigma T6791, store at room temperature
TRIS HCl - Sigma T3253, store at room temperature

Solutions:

I. TRIS BUFFER 0.05 M, pH 7.6 @ room temperature
TRIS HCl 1.43 g
TRIS BASE 0.415 g
Deionized water 250 ml: Store refrigerated, prepare every four to six weeks

II. NADH SOLUTION (8 mg/5 ml)
NADH 80 mg
TRIS BUFFER (solution I) 50 ml
Dispense 5 ml aliquots into 13x100 mm glass tubes
Cover with caps
Store at -20 oC

III. NBT SOLUTION (10 mg/5ml)
Nitro-Blue Tetrazolium 200 mg
TRIS BUFFER (solution I) 100 ml
Store refrigerated in a glass reagent bottle
Prepare every four to six weeks

IV. ACETONE DESTAINING SOLUTIONS (30%, 60%, 90%): These solutions need only be approximate
30 % ACETONE 10 ml + Deionized water 20 ml
60 % ACETONE 20 ml + Deionized water 10 ml
90 % ACETONE 30 ml

V. Aqueous Mounting Medium (glycerogel)
Gelatin ( ICN#960317 - 100 bloom 4 g
Glycerol 25 ml
Phenol (CAUSTIC !) 0.5 ml
Deionized water 21 ml

1. Dissolve gelatin in boiling water.

2. Cool, but do not allow to solidify.

3. Add phenol and glycerol.

4. Mix well.

5. Allow air bubbles in mixture to dissipate before using!

Staining Procedure:

1. Thaw NADH solution.

2. Add 5 ml NBT solution to the tube with the NADH solution.

3. Incubate coverslips in a columbia staining dish (Thomas Scientific #8542-C12) for 30 minutes at 37 oC.

4. Wash with three exchanges of tap or deionized H2O.

5. Remove unbound NBT from the sections with three exchanges each of the 30, 60 and 90 % acetone solutions in increasing then decreasing concentration. Leave the 90 % acetone covering the sections until a faint purplish cloud is seen over the section.

6. Finally, rinse several times with deionized H2O and then mount the coverslips with the aqueous mounting medium onto a labeled glass slide.

Results:

Purple formazan precipitate is deposited at sites of mitochondria in sarcoplasmic network. Type I fibers are darker than those of type II. Walls of blood vessels also are stained.

REFERENCES:

1. Sheehan, D.C. and Hrapchak, B.B.: THEORY AND PRACTICE OF HISTOTECHNOLOGY Second Edition, Battelle Memorial Institute, 1987.


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6/15/98