CLINICAL LABORATORY
PRINCIPLE:
In the histochemical reaction, phosphorylase acts on the substrate, glucose-1-phosphate and forms, in the presence of a glycogen primer, a polysaccharide composed of -1,4-glycosyl units. The in vitro reaction of polysaccharide formation is, therefore, the opposite of the in vivo action of glycogen degradation. This happens because the concentration of the substrate is high, and the concentration of the inorganic phosphate is low. The system equilibrium therefore favors glycogen formation. Adenosine-5'-monophosphate functions as an activator.
Exposure of the sections to an iodine solution
after incubation results in a varied color formation in the newly
formed polysaccharide. A negative reaction is yellow, and it
has been shown that unbranched chains of 4 to 6 glucosyl units
will give a negative reaction. A polysaccharide of 8 to 12 units
gives a reddish color, followed by various transitional colors
as the length of the chain increases. Chain lengths of 30 to
35 units give a blue color. The reason the color is blue rather
than red-brown is that the polysaccharide formed by the phosphorylase
action is not normal glycogen. For glycogen to color a true red-brown,
it has to be branched, and branching will only occur if branching
enzyme is allowed to act. However, the action of the branching
enzyme is eliminated by the inclusion of alcohol in the incubation
medium.
QUALITY ASSURANCE:
This enzyme is very labile and the preliminary
handling of the specimen is of the utmost importance. Biopsies
that have been totally immersed in saline for any length of time
or removed with a cautery knife are usually badly compromised.
However, there will most often be some areas of stain as opposed
to the diffuse yellow color of a true negative stain.
SPECIMEN REQUIRED:
Snap frozen human striated muscle. (Use the
isopentane freezing method described elsewhere.)
Controls:
Use skeletal muscle for a positive control.
Omit the glucose-1-phosphate for a negative control.
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
- Columbia staining dish - Thomas Scientific #8542-C12
- Reagents:
- Absolute alcohol ( 100% ethanol) - Quantum,
FLAMMABLE store at room temp. in a flammable cabinet
- Acetic Acid (glacial acetic acid Fisher Scientific A507-500, store at room temp.)
- Adenosine Monophosphate (Adenylic Acid, AMP) (Sigma A 1752, sodium salt; store at -20 C, desiccated)
- Glucose-1-Phosphate (Sigma G 7000; store at -20 C, desiccated)
- Glycerol (Sigma G 8773, store at room temp.)
- Glycogen (Sigma G 4011, from rabbit liver, store at 0-5 C, desiccated)
- Potassium Iodide (Sigma P 8256, TERATOGEN, store at room temp.)
- Reagent alcohol, ACS - histological Fisher A962-4,or HPLC A995 FLAMMABLE, TOXIC, TERATOGENIC, store at room temp. in flammable cabinet
- Sodium acetate (Sigma S 9513, trihydrate, store at room temp.)
- Sodium Hydroxide (Certified ACS pellets - Fisher S318, CAUTION CORROSIVE!!)
- Acetic Acid (glacial acetic acid Fisher Scientific A507-500, store at room temp.)
Solutions:
- I. Glucose-1-Phosphate, 1% (w/v)
- Glucose-1-phosphate 500 mg
- Deionized water 50 ml
- Store at -20 C in 2.5 ml aliquots
- Deionized water 50 ml
- II. Adenosine Monophosphate, 0.2% (w/v)
- Adenosine monophosphate 100 mg
- Deionized water 50 ml
- Store at -20 C in 2.5 ml aliquots
- Deionized water 50 ml
- III. Sodium Acetate Solution, 0.1 M
- Sodium acetate CH3COONa3H2O 2.72 g
- Deionized water 200 ml
- Deionized water 200 ml
- IV. Acetic Acid, 0.1N
- CH3COOH 0.575 ml
- Add to deionized water 100 ml
- Add to deionized water 100 ml
- IV. Sodium Acetate Buffer, pH 5.6, 0.1 M
- Solution III: 181 ml
- Solution IV: 19 ml
- Solution IV: 19 ml
- V. Glycogen, 0.02%
- Glycogen 20 mg
- Solution IV 100 ml
- Store at -20 C in 5 ml aliquots
- Solution IV 100 ml
- VI. Ethanol, 95 % (v/v)
- Reagent alcohol 95 ml
- Deionized water 5 ml
- Deionized water 5 ml
- VII. Lugol's Iodine Solution: (store at room temp. in an amber bottle)
- Potassium Iodide (KI) 1 g
- Dissolve in deionized water 1 ml
- When completely dissolved add iodine crystals (I2) 0.5 g
- When iodine is completely dissolved add D.I. H2O to a final volume 25 ml
- Dissolve in deionized water 1 ml
- VIII. Working Solution for Stain (prepare fresh for each assay)
- Thaw ahead and bring to room temperature
- To a 20 ml glass beaker add solutions I, II, V
- Add 1.5 ml 100 %(absolute) Ethanol
- Adjust pH to 5.6
- To a 20 ml glass beaker add solutions I, II, V
- Staining Procedure:
- 1. Place coverslips with sections in a columbia
staining dish (Thomas Scientific #8542-C12) for 5 minutes at room temperature
for a minimum of 45 minutes (overnight is very acceptable).
- 2. Wash with several exchanges of deionized H2O.
- 3. Add 6 - 8 drops of Lugol's Iodine to approx. 10 ml deionized water.
- 4. Add iodine solution to sections until they are dark in color.
- 5. Wipe the back of the coverslip with a cotton tipped swab to remove excess iodine solution.
- 6. Mount coverslip onto a labeled glass slide with glycerol that has been colored with a few drops of Lugol's Iodine.
- 2. Wash with several exchanges of deionized H2O.
Results:
Sites of phosphorylase activity -- blue or
reddish brown deposits of newly synthesized polysaccharide.
REFERENCES:
1. Thompson, Samuel W. SELECTED HISTOCHEMICAL
AND HISTOPATHOLOGICAL METHODS, Charles C. Thomas, Springfield,
IL, 1966.
2. Sheehan, D.C. and Hrapchak, B.B., THEORY
AND PRACTICE OF HISTOTECHNOLOGY, 2nd Edition; Battelle Memorial
Institute, Columbus, OH, 1987.
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8/14/97