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Periodic Acid Schiff Protocol
This type of staining is used for the detection of glycogen. Tissue sections are first oxidized by 0.5% periodic acid solution. The oxidative process results in the formation of aldehyde groupings through carbon-to-carbon bond cleavage. Free hydroxyl groups should be present for oxidation to take place. Oxidation is completed when it reaches the aldehyde and the aldehyde groups are detected by the Schiff reagent. A colorless, unstable dialdehyde compound is formed and then transforms to the colored final product through the restoration of the quinoid chromophoric grouping.
The PAS stain, along with diastase or amylase digestion has histochemical specificity for glycogen. Skeletal muscle normally contains glycogen and is often recommended as a positive control tissue.
Snap-frozen human striated muscle.
From the snap-frozen biopsy, cut 10-16 micron (12 µm) sections in a crytostat. Place one or more sections to a No.1, 22 mm square coverslip.
- Ceramic staining rack – Thomas Scientific #8542-E40
- Columbia staining dish – Thomas Scientific #8542-C12
- Columbia staining dish (jar) – Thomas Scientific #8542-E30
- Latex gloves
- Absolute alcohol (100% ethanol) – Quantum, FLAMMABLE store in flammable cabinet at room temperature
- Glacial Acetic Acid -Fisher A507-500, CORROSIVE store at room temperature
- Amylase – Sigma A-6505, store at room temperature
- Chloroform – Baxter 049-4, FLAMMABLE CARCINOGEN, store in a flammable cabinet at room temperature)
- Periodic Acid – Sigma P7875, store at room temperature
- Permount – Fisher SP15-100, FLAMMABLE HEALTH HAZARD
- Reagent alcohol, ACS – histological Fisher A962-4 or HPLC A995, FLAMMABLE, TOXIC, TERATOGENIC, store in flammable cabinet at room temperature
- Schiff Reagent – Harleco 6073/71, store at room temperature
- Xylenes – Fisher #HC700-1GAL, FLAMMABLE, store in flammable cabinet at room temperature
Carnoy’s Fixative (store at room temperature) PREPARE IN A FUME HOOD
100% Alcohol ~ 60 ml
Chloroform ~ 30 ml
Glacial acetic acid ~10 ml
Periodic Acid Solution, 0.5 % (w/v) PREPARE FRESH FOR EACH STAIN
Periodic acid ~ 50 mg
Dissolved in deionized water ~ 10 ml
Reagent alcohol ~ 50 ml
Deionized water ~ 50 ml
Reagent alcohol ~ 70 ml
Deionized water ~ 30 ml
Reagent alcohol ~ 80 ml
Deionized water ~ 20 ml
Reagent alcohol ~ 95 ml
Deionized water ~ 5 ml
- Place the coverslip with the section on it into a columbia staining dish (Thomas Scientific (#8542- E40).
- Add enough Carnoy’s fixative to cover the coverslip and incubate for 10 minutes. (Specific Volume? Enough to cover section?)
- Very carefully, rinse the (coverslip, dish, section?) with several exchanges of deionized water. Sections may wash off!!
- Add enough Periodic Acid solution to cover the coverslip and incubate for 10 minutes.
- Very carefully, rinse the coverslip with several exchanges of deionized water. Sections may wash off!! Add enough Shiff Reagent to cover the coverslip and incubate for 5 minutes
- Carefully wash with three exchanges of tap or deionized H2O.
- Dehydrate coverslip in ascending alcohol solutions (50%, 70%, 80%, 95% x 2, 100% x 2) in columbia staining dish(jar)es – Thomas Scientific #8542-E30.
- Rinse with xylene (3 – 4 x ) also in columbia staining dish(jar) – Thomas Scientific #8542-E30.
- Mount coverslip onto a labeled glass slide with Permount or any suitable organic mounting medium
Glycogen, neutral mucosubstances, basement membranes, collagen fibers, glycolipids and phospholipids will be demonstrated through a range of pink, red or purple colors. If diastase or amylase is used as a negative control, the glycogen deposits are removed leaving only the plasma membrane, which will stain pink. The two major types of fibers are usually distinguished by different intensities of staining.
- Thompson, Samuel W. SELECTED HISTOCHEMICAL AND HISTOPATHOLOGICAL METHODS, Charles C. Thomas, Springfield, IL, 1966
- Sheehan, D.C. and Hrapchak, B.B., THEORY AND PRACTICE OF HISTOTECHNOLOGY, 2nd Edition; Battelle Memorial Institute, Columbus, OH, 1987