CLINICAL LABORATORY
PRINCIPLE:
This is a
generic name for phosphomonoesterases that hydrolyze orthophosphate
at an alkaline pH. These enzymes are widely distributed and usually
activated by magnesium, manganese, zinc, and cobalt ions and are
inhibited by cysteine, cyanides, and 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 and 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 muscle. (Use the isopentane freezing method described elsewhere.)
Controls:
Small arterioles (< 80 μm diameter) and some capillaries
stain 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 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:
Glacial Acetic Acid -Fisher A507-500, CORROSIVE store at room temp.
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 SUBSTANCE,
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 stain)
10 ml 0.1 M Barbital Solution
20 mg Sodium α-Napthyl Acid Phosphate
10 mg Fast Blue RR salt (a fine brown precipitate will form)
Adjust pH to 9.2 with 1 N NaOH (1 - 2 drops)
Filter solution just prior to use
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. Place in 1 % Acetic Acid for 10 minutes.
4. Rinse with deionized water, 2 to 3 changes.
5. Let air-dry for at least one hour (overnight
is better).
6. Rehydrate with D.I. water (approximately
10 minutes).
7. Clean back of coverslips with cotton swab.
5. Mount with aqueous medium (e.g. glycerogel).
Results
Sites of alkaline phosphatase activity are
localized as a fine precipitate colored dark-blue/black.
REFERENCES
1. Barka and Anderson, HISTOCHEMISTRY,
Harper & Row, New York, 1963.
2. Sheehan and Hrapchak, HISTOTECHNOLOGY, 2nd Edition; Batelle Press, Columbus, 1987.
3. Thompson, SELECTED HISTOCHEMICAL
AND HISTOPATHOLOGICAL METHODS, Charles C. Thomas, Springfield,
IL, 1966.
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05/27/2000