Glycogen Phosphorylase Stain

Solutions

1. 0.1M Acetate/Sodium Acetate Buffer

2.97 g Sodium Acetate

0.22ml Glacial Acetic Acid

400 ml Distlled H₂O

Adjust pH to 5.6

2. Incubation Medium

40 ml 0.1M Acetate/Sodium Acetate Buffer

140 ml distilled H₂O

400 mg EDTA

320 mg Sodium Fluoride

8 g Dextran (MW~40,000; Sigma prod. D-4133)

1.6 gm alpha-D-Glucose-1-Phosphate

(For total GPase(a+b))

160 mg AMP

Adjust pH to 6.0

Make up to 200 ml with distilled H₂O

3. Lugol's Iodine

2 gm Potassium Iodide (KI)

1 gm Iodine

300 ml distilled H₂0

33 gm Sucrose

Dissolve KI in H₂0 then add Iodine until dissolved. Finally add sucrose. Stir well.

Store in the dark.

Tissue Processing

1. Cut 20 - 30 micron sections
2. Air dry 30 - 40 minutes
3. Incubate 30 minutes in Medium at 37° C.
4. Air dry 20 minutes
5. Fix tissue in 40% Ethanol for 4 minutes.
6. Air dry 10 minutes.
7. Place in Lugol's Iodine 2-5 minutes
8. Wash in normal saline
9. Air dry 30 minutes
10. Coverslip

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Steve Milway

Last modified: Wednesday, December 17, 2003