"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.
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.
- Fixation: None, use snap frozen tissue.
- Technique: Cut 10 - 16 micron (12 µm)
sections in cryostat from snap frozen biopsy. Attach one or
more sections to a No. 1½, 22 mm square coverslip.
staining rack - Thomas Scientific #8542-E40
staining dish - Thomas Scientific #8542-C12
- Columbia staining dish(jar) - Thomas Scientific
- Latex gloves
- Acetone - Baxter #010-4, FLAMMABLE
- Deionized water
- Gelatin - 100 bloom -ICN 960317 store at
- Glycerol -Sigma G 8773, store at room temperature
- 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
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
- 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.
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
1. Sheehan, D.C. and Hrapchak, B.B.: THEORY
AND PRACTICE OF HISTOTECHNOLOGY Second Edition, Battelle
Memorial Institute, 1987.