CLINICAL LABORATORY

CYTOCHROME OXIDASE STAINING PROTOCOL

PRINCIPLE:

Cytochrome oxidase is the collective name for part of the oxidative respiratory chain of enzymes located exclusively in the mitochondria of cells. Sometimes cytochrome oxidase was regarded as synonymous with cytochrome a3. This method is a modification of the "Nadi" reaction. The use of 3,3' diaminobenzidine (DAB) results in a brown insoluble compound at the site of cytochrome oxidase activity.

SPECIMEN REQUIRED:

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

Controls: Use myocardium for a positive control. For a negative control, cyanide or sodium azide inhibits cytochrome oxidase activity.

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.1 1/2, 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:

Catalase (Sigma C-10)

Cytochrome C (Sigma C-2506)

Deionized water

3, 3' diaminobenzidine tetrahydrochloride (Sigma D-5637)
CARCINOGENIC, store at -20°C

Permount - Fisher SP15-100, FLAMMABLE HEALTH HAZARD

Reagent alcohol, ACS histochemical Fisher A962-4,or HPLC A995
FLAMMABLE, TOXIC, store at room temp. in flammable cabinet

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)hexptahydrate (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

Sucrose - Sigma S7903, store at room temperature

Xylenes - Fisher #HC700-1GAL, FLAMMABLE, store room temperature in flammable cabinet

Solutions:

I. 0.2 M Phosphate Buffer, pH 7.6

• 0.2 M sodium dibasic phosphate (Na2HPO4)heptahydrate (53.65 gm/liter deionized H2O) or anhydrous (28.39gm/liter) 87 ml
• 0.2 M sodium monobasic phosphate (NaH2PO4) monohydrate (27.8 gm/liter deionized H2O) 13 ml

II. Incubating Solution:

• dissolve 750 mg Sucrose in 7.5 ml deionized H₂O
• add 2.5 ml 0.2 M Phosphate Buffer
• add 5 mg DAB and dissolve
• add 10 mg cytochrome C
• add 20 μg catalase (a few crystals)

III. Alcohol 50 % (v/v)

• Reagent alcohol ~50 ml
• Deionized water ~50 ml

IV. Alcohol 70 %(v/v)

• Reagent alcohol ~70 ml
• Deionized water ~30 ml

V. Alcohol 80 % (v/v)

• Reagent alcohol ~80 ml
• Deionized water ~20 ml

VI. Alcohol 95 % (v/v)

• Reagent alcohol ~95 ml
• Deionized water ~5 ml

Staining Procedure

1. Place coverslips in the incubating solution in a columbia staining dish (Thomas Scientific #8542-C12) for 60 minutes at room temperature.

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

3. Dehydrate in ascending alcohols (50%,70%,80%,95% x 2, 100% x 2) in columbia staining dish(jar) - Thomas Scientific #8542-E30.

4. Clear with at least 2 changes of xylene columbia staining dish(jar) - Thomas Scientific #8542-E30

5. Mount with PERMOUNT or another synthetic organic mounting medium.

Results

Sites of cytochrome oxidase activity are colored brown.

REFERENCE

1. Seligman et al., 1968. In: HISTOCHEMISTRY - THEORETICAL and APPLIED, A.G. Everson Pearse, Vol. 2, 3rd Edition; Williams & Wilkins, Baltimore, 1972.

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7/10/98