Home, grave, Indice alfabetico, Collegamenti, Patologia, Molecole, Sindromi, Muscoli, Giunzioni neuromuscolari, Nervi, Spinale, Atassia, Anticorpi e Biopsia, Informazioni per i pazienti TRADUZIONE di Natale Marzari

PRINCIPLE:

The complex naphthol, naphthol acid phosphate, is hydrolyzed by acid phosphatases presenti in the tissue, e napthol derivatives sono therby produced. The naphthol derivatives couple con 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 muscoli. (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 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:

Calcio 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,- istochimiche Fisher A962-4 o 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 SOSTANZA, 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. e 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)

Calcio 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 e 6.0 con alkali

VII. Alcol 50 %

reagent alcohol ~50 ml

deionized water ~50 ml

VIII. Alcol 70 %

reagent alcohol ~70 ml

deionized water ~30 ml

IX. Alcol 80 %

reagent alcohol ~80 ml

deionized water ~20 ml

X. Alcol 95 %

reagent alcohol ~95 ml

deionized water ~ 5 ml

Colorazioneing Procedure:

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

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

3. Add incubation solution e Colorazione for at least one (1) ore at room temperature in a dark place.

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

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

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

7. Mount con PERMOUNT o another synthetic organic mounting medium.

Results:

A red azo dye indicates sites of acid phosphatase activity.

REFERENCES:

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

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

PREPARED BY	G. JAMES PLANER	07/22/85
SUPERCEDES REVISION	G. JAMES PLANER	04/27/94
REVISED BY	KAREN BIESER	07/18/97
ADOPTED BY	ALAN PESTRONK, M.D.
REVIEWED BY
REVIEWED BY
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