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Dopo 41 anni e 5 mesi, nel maggio 2006 la magistratura di Trento ha riconosciuto l'esistenza e la gravità di quella malattia rara che nessuna altra istituzione o persona singola della provincia di Trento ancora mi riconosce, e per negare la quale mi perseguita.  Natale Marzari

PRINCIPLE:

This is a generic name for phosphomonoesterases that hydrolyze orthophosphate at an alkaline pH. These enzymes sono widely distributed e usually activated by magnesium, manganese, zinc, e cobalt ions e sono inhibited by cysteine, cyanides, e arsenates. This is a simultaneous coupling azo dye method first developed in 1944. It has been modified repeatedly since that time. Sodium -naphthyl acid phosphate is the substrate used in this protocol which is hydrolyzed e then coupled to the diazonium salt (Fast Blue RR) which is then precipitated at the site of enzyme activity.

SPECIMEN REQUIRED:

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

Controls:

Sottili arterioles (< 80 μm diameter) e some capillaries Colorazione positively.
For a negative control, leave out the 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:

  Glacial Acetic Acid -Fisher A507-500, CORROSIVE store a temperatura ambiente.

  Fast Blue RR salt - Sigma F0500 store at -20 desiccated

  α-Napthyl acid phosphate, monosodium salt (C₁₀H₈O₄P Na)- Sigma N7000

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

Solutions:

I. 0.1 M Sodium Barbital Solution (5.15 gm barbital powder (room temp.) + deionized H2O 250 ml)

II. 1 % Acetic Acid (1 ml glacial acetic acid to deionized H₂O 100 ml)

III. 1 N NaOH

IV. Incubating Solution (prepare fresh for each Colorazione)

  10 ml 0.1 M Barbital Solution

  20 mg Sodium α-Napthyl Acid Fosfato

  10 mg Fast Blue RR salt (a fine brown precipitate will Form)

Adjust pH to 9.2 con 1 N NaOH (1 - 2 drops)

Filter solution just prior to use

Colorazioneing Procedure

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

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

3. Place in 1 % Acetic Acid for 10 minutes.

4. Rinse con deionized water, 2 to 3 changes.

5. Let air-dry for at least one ore (overnight is better).

6. Rehydrate con D.I. water (approximately 10 minutes).

7. Clean back of coverslips con cotton swab.

5. Mount con aqueous medium (e.g. glycerogel).

Results

Sites of alkaline phosphatase activity sono localized as a fine precipitate colored dark-blue/black.

REFERENCES

1. Barka e Anderson, HISTOCHEMISTRY, Harper e Row, New York, 1963.

2. Sheehan e Hrapchak, HISTOTECHNOLOGY, 2a Edition; Batelle Press, Columbus, 1987.

3. Thompson, SELECTED HISTOChimici AND HISTOPATHOLOGICAL METHODS, Charles C. Thomas, Springfield, IL, 1966.

PREPARED BY	G. JAMES PLANER	10/22/90
SUPERCEDES REVISION	G. PLANER	12/14/95
REVISED BY	KAREN BIESER	7/14/97
ADOPTED BY	ALAN PESTRONK, M.D.	7/14/97
REVIEWED BY	ALAN PESTRONK, M.D.	9/29/99
REPLACED

05/27/2000