Técnicas Histoenzimáticas - Princípios Teóricos

Miofosforilase

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Solutions:
VI. Ethanol, 95 % (v/v)
I. Glucose-1-Phosphate, 1% (w/v)
Glucose-1-phosphate 500 mg
VII. Lugol's Iodine Solution: (store at
room temp. in an amber bottle)
II. Adenosine Monophosphate, 0.2% (w/v)
Potassium Iodide (KI) 1 g
Adenosine monophosphate 100 mg
Dissolve in deionized water 1
ml When completely
III. Sodium Acetate Solution, 0.1 M
dissolved add iodine crystals
Sodium acetate CH3COONa3H2O
(I2) 0.5 g
2.72 g
When iodine is completely
Deionized water 200 ml
dissolved add D.I. H2O to a
final volume 25 ml
IV. Acetic Acid, 0.1N
CH3COOH 0.575 ml
VIII. Working Solution for Stain
Add to deionized water 100 ml
(prepare fresh for each assay)
Thaw ahead and bring to
IV. Sodium Acetate Buffer, pH 5.6, 0.1 M
room temperature
Solution III: 181 ml
To a 20 ml glass beaker add
Solution IV: 19 ml
solutions I, II, V
Add 1.5 ml 100 %(absolute)
V. Glycogen, 0.02%
Ethanol
Glycogen 20 mg
Adjust pH to 5.6
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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.

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 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.

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 - 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.

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 - 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

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 - 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

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