CLINICAL LABORATORY

SUCCINIC DEHYDROGENASE PROTOCOL

PRINCIPLE:

Succinic dehydrogenase (SDH), is a soluble iron flavoprotein that catalyzes the reversible oxidation of succinic acid to fumaric acid. The histochemical demonstration of the activity of this enzyme is achieved by incubation of fresh frozen sections with a succinate substrate in the presence of a tetrazolium compound. Tetrazoliums are water-soluble compounds employed in histochemistry as redox indicators. Under appropriate conditions, tetrazoliums are reduced to formazans which are water-insoluble tetrazolium (NBT). Enzymatic activity releases hydrogen from colored compounds. Commonly used tetrazoliums include nitro blue 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 described elsewhere.)

Controls: Use myocardium for a positive control. For a negative control, eliminate sodium succinate from the incubating medium.

METHOD:

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.

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

Nitro blue tetrazolium - Sigma N6876 store desiccated at 0 - 5 ^o C

Phenol - Fisher A931-1, CAUSTIC, Store at room temperature

Sodium dibasic phosphate (Na₂HPO₄) anhydrous, ACS (FW 141.96)-Sigma S9763 or Fisher S374, or Mallinckrodt 7917; Store at room temperature

Sodium dibasic phosphate (Na₂HPO₄) heptahydrate (FW 268.07) Sigma S9390, or Fisher S373; Store at room temperature

Sodium monobasic phosphate (NaH₂PO₄) monohydrate (FW 137.99) , ACS - Sigma 9638 or Fisher S369, or Mallinckrodt 7892; Store at room temperature

Succinic acid,disodium salt - Sigma S2378, store at room temperature

Solutions:

I. 0.2 M Phosphate Buffer, pH 7.6

0.2 M Sodium monobasic phosphate (NaH₂PO₄) 13 ml (27.8 gm/liter deionized H₂O)

0.2 M Sodium dibasic phosphate (Na₂HPO₄) heptahydrate (53.65 gm/liter deionized H2O) OR Sodium dibasic phosphate (Na₂HPO₄) anhydrous (28.39g/liter deionized water) 87 ml

II. 0.2 M Succinic Acid (sodium salt) with deionized H2O 5.4 g/100ml

It is recommended to prepare FRESH each time it is needed .

A stock solution may be kept refrigerated for two weeks and still be effective.

III. 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. Prepare the incubation medium as follows:

0.2 M phosphate buffer 10 ml

Dissolve Sodium Succinate 270 mg & NBT 10 mg

2. Incubate coverslips in a Columbia staining dish (Thomas Scientific #8542-C12) for 60 minutes at 37 ^oC.

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

4. Prepare approximate solutions of 30, 60 and 90 % acetone using deionized H₂O and remove unbound NBT from the sections with three exchanges each of the acetone solutions in increasing then decreasing concentration. Leave the 90 % acetone covering the sections until a faint purplish cloud is seen over the section.

5. Finally, rinse several times with deionized H2O and then mount the coverslips with the aqueous mounting medium.

Results:

Purple formazan precipitate is deposited at sites of mitochondria in sarcoplasmic network. Type I fibers are darker than those of type II. Results appear similar to those of the NADH stain but not as intense. Walls of blood vessels also are stained. Best results occur if the sections are stained on the same day that they are cut.

REFERENCES:

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

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8/14/97