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PRINCIPLE:

Succinic deidrogenasi (SDH), is a soluble iron flavoprotein that catalyzes the reversible oxidation of succinic acid to fumaric acid. The istochimiche demonstration of the activity of this enzyme is achieved by incubation of fresh frozen sections con a succinate substrate in the presence of a tetrazolium compound. Tetrazoliums sono water-soluble compounds employed in histochemistry as redox indicators. Under appropriate conditions, tetrazoliums sono ridotta to Formazans which sono water-insoluble tetrazolium (NBT). Enzymatic activity releases hydrogen from colored compounds. Commonly used tetrazoliums include nitro blue the substrate, e 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 muscoli. (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 Biopsia. Attach one o più sections to a No. 1½, 22 mm squsono coverslip.

Equipment:

Ceramic Colorazioneing rack - Thomas Scientific #8542-E40

Columbia Colorazioneing dish - Thomas Scientific #8542-C12

Columbia Colorazioneing 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 o Fisher S374, o Mallinckrodt 7917; Store at room temperature

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

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

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

Solutions:

I. 0.2 M Fosfato 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) con 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 settimane e 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 e glycerol.

4. Mix well.

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

Colorazioneing Procedure:

1. Prepare the incubation medium as follows:

0.2 M phosphate buffer 10 ml

Dissolve Sodium Succinate 270 mg e NBT 10 mg

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

3. Wash con three exchanges of tap o deionized H2O.

4. Prepare approximate solutions of 30, 60 e 90 % acetone usando deionized H₂O e remove unbound NBT from the sections con 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 numerosi times con deionized H2O e then mount the coverslips con the aqueous mounting medium.

Results:

Purple Formazan precipitate is deposited at sites of mitocondri in sarcoplasmic network. Tipo I fibre sono darker than those of tipo II. Results appear similar to those of the NADH Colorazione ma non as intense. Walls of blood vessels also sono Colorazioneed. Best results occur if the sections sono Colorazioneed on the same day that they sono cut.

REFERENCES:

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

PREPARED BY	G. JAMES PLANER	10/22/90
SUPERCEDES REVISION	G. JAMES PLANER	04/27/94
REVISED BY	KAREN BIESER	07/17/97
ADOPTED BY	ALAN PESTRONK, M.D.
REVIEWED BY
REVIEWED BY
REPLACED

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8/14/97