CLINICAL LABORATORY
PERIODIC ACID SCHIFF (PAS) PROTOCOL
IMAGE
PRINCIPLE:
This stain is used for the demonstration of
glycogen. Tissue sections are first oxidized by periodic acid.
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 stage. The aldehyde groups are detected
by the Schiff reagent. A colorless, unstable dialdehyde compound
is formed and then transformed to the colored final product by
restoration of the quinoid chromophoric grouping.
QUALITY ASSURANCE:
The PAS stain with diastase or -amylase digestion
has histochemical specificity for glycogen. Skeletal muscle normally
contains glycogen and is often recommended as a positive control
tissue.
SPECIMEN REQUIRED:
Snap frozen human striated muscle. (Use the
isopentane freezing method previously described.)
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:
- Absolute alcohol (100%
ethanol) - Quantum, FLAMMABLE store at room temp.
in a flammable cabinet
- 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 at room temperature in a flammable
cabinet)
- 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
at room temperature In flammable cabinet
- Schiff Reagent - Harleco 6073/71, store at
room temperature
- Xylenes - Fisher #HC700-1GAL, FLAMMABLE,
store room temperature in flammable cabinet)
Solutions:
- I. Carnoy's Fixative (store at room temperature) PREPARE IN A FUME HOOD
- Alcohol, 100 % 60 ml
- Chloroform 30 ml
- Glacial acetic acid 10 ml
- II. Periodic Acid Solution, 0.5 % (w/v) PREPARE FRESH FOR EACH STAIN
- Periodic acid 50 mg dissolved in deionized water 10 ml
- III. Alcohol 50 %
- Reagent alcohol ~50 ml
- Deionized water ~50 ml
- IV. Alcohol 70 %
- Reagent alcohol ~70 ml
- Deionized water ~30 ml
- V. Alcohol 80 %
- Reagent alcohol ~80 ml
- Deionized water ~20 ml
- VI. Alcohol 95 %
- Reagent alcohol ~95 ml
- Deionized water ~ 5 ml
Staining Procedure:
- Place the coverslip with section in a columbia
staining dish (Thomas Scientific #8542- E40).
- Add Carnoy's fixative to dish for 10 minutes.
- Rinse very carefully with several exchanges
of deionized water. Sections may wash off!!
- Add Periodic Acid solution to staining
dish for 10 minutes.
- Rinse very carefully with several exchanges
of deionized water. Sections may wash off!!
- Add Shiff Reagent for 5 minutes
- Carefully wash with three exchanges of
tap or deionized H2O.
- Dehydrate in ascending alcohol solutions
(50%, 70%, 80%, 95% x 2, 100% x 2) in columbia staining dish(jar)s
- Thomas Scientific #8542-E30.
- Clear 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 some other suitable organic mounting medium
Results:
Glycogen, neutral mucosubstances, basement
membranes, collagen fibers, glycolipids and phospholipids will
be demonstrated as pink to red to purple color.
If diastase or -amylase is used for a negative
control, the glycogen deposits are removed leaving only the plasma
membrane staining pink. The two major types of fibers are usually distinguished by different
intensity of staining.
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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7/9/2008