PRINCIPLE:

The complex naphthol, naphthol acid phosphate, is hydrolyzed by acid phosphatases present in the tissue, and napthol derivatives are therby produced. The naphthol derivatives couple with the unstable diazonium salt, hexazonium pararosanilin, to produce a red azo dye to mark the site of enzyme activity.

SPECIMEN REQUIRED:

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

Controls:

Use prostate gland as a positive control. For a negative control, eliminate the enzymatic substrate.

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:

Calcium Chloride, anhydrous - Sigma C4901, store at room temperature

Formaldehyde, 37 % - Fisher F79-500, POISON, CARCINOGEN, store at room temperature

hydrochloric acid, ACS - Fisher A144-500, CORROSIVE, store at room temperature

α-napthyl acid phosphate, monosodium salt (C₁₀H₈O₄PNa)- Sigma N7000

pararosaniline hydrochloride certified - Sigma P1528, store at room temperature

Permount - Fisher SP15-100, FLAMMABLE HEALTH HAZARD

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

Sodium acetate, trihydrate - Sigma S9513, store at room temperature

sodium barbital (5,5' dietyl barbituric acid) - Sigma B0500, NARCOTIC, TOXIC,CONTROLLED SUBSTANCE, store at room temperature

sodium nitrite certified crystalline - Fisher S347 - STRONG OXIDIZER, COMBUSTIBLE

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

I. Barbital Acetate Solution

sodium barbital(sodium barbiturate) 1.47 g

sodium acetate(NaC2H3O2.3H2O) 0.97 g

deionized water -> final volume of 50 ml

II. Pararosaniline HCl Solution

Pararosaniline Hydrochloride (C.I. 42500) 0.5 g

deionized water 10.0 ml

Concentrated Hydrochloric Acid 2.0 ml

dissolve dye in water, add acid, heat gently, cool

to room temp. and filter

Store at 4oC.

III. Sodium Nitrite, 4% (w/v)

Sodium Nitrite (NaNO2) 0.4 g

deionized water 10.0 ml

IV. Baker's Solution (modified)

Calcium Chloride (anhydrous) CaCl2 0.3 g

Formaldehyde, 37% 3.0 ml

deionized water -> final volume of 100 ml

V. Incubating Solution:

Sodium alpha napthyl acid phosphate 20 mg

Barbital Acetate Solution 5.0 ml

deionized water 13.0 ml

VI. Indicator Solution:

Pararosaniline HCl Solution 0.8 ml

Sodium Nitrite Solution, 4% 0.8 ml

Mix well, let stand for 2 minutes, then add to Incubating Solution

Adjust pH to between 5.0 and 6.0 with alkali

VII. Alcohol 50 %

reagent alcohol ~50 ml

deionized water ~50 ml

VIII. Alcohol 70 %

reagent alcohol ~70 ml

deionized water ~30 ml

IX. Alcohol 80 %

reagent alcohol ~80 ml

deionized water ~20 ml

X. Alcohol 95 %

reagent alcohol ~95 ml

deionized water ~ 5 ml

Staining Procedure:

1. Place coverslips with sections in Baker's Solution in a columbia staining dish (Thomas Scientific #8542-C12) for 5 minutes at room temperature.

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

3. Add incubation solution and stain for at least one (1) hour at room temperature in a dark place.

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

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

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

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

Results:

A red azo dye indicates sites of acid phosphatase activity.

REFERENCES:

1. Barka, T., 1961. In: SELECTED HISTOCHEMICAL AND HISTOPATHOLOGICAL METHODS, S.W. Thompson; Charles C. Thomas, Springfield, IL, 1966.

2. Barka, T. and Anderson, P.J., 1962. In: THEORY AND PRACTICE OF HISTOTECHNOLOGY, Sheehan, D.C. and Hrapchak, B.B., 2nd Edition 1980; Battelle Memorial Institute, Columbus, OH, 1987.