Periodic Acid-Schiff (PAS), +/- Diastase Digestion
Purpose
PAS: To determine the presence of PAS positive substances, such as glycogen, cellulose, starch, basement membrane, neutral mucins, and chitin.
PAS with diastase digestion: To determine the presence of glycogen by first digesting it out of tissue sections and subsequently staining with PAS stain.
Principle
The diastase reaction
Alpha-amylase, also known as diastase, is an enzyme commonly present in saliva. The diastase acts on glycogen to depolymerize it into smaller sugar units, maltose and glucose, that are washed out of the section.
The PAS reaction
Periodic acid (HIO4) solutions oxidise 1,2-glycol groups, producing a dialdehyde. Schiff reagent is prepared by treating basic fuchsin (parasoaniline) with sulphurous acid. Reduction (breaking of the double bond) causes loss of the quinoid structure and “masking” of the chromophores and the formation of a colorless compound called “leucofuchsin” (not a true leucofuchsin but referred to as this by most).
Following the Schiff reaction washing in running water causes the loss of the bound sulphurous acid group attached to the central carbon atom, the restoration of the quinoid structure in the dye bound by the aldehyde and the colour precipitation of an insoluble magenta coloured complex (aldehyde fuchsin).
Solutions
Digestion Solution
- Diastase (with alpha-amylase) 0.05 g
- Distilled water 50.0 ml
Make fresh, use within 15 minutes, discard.
**Saliva can be substituted (mouth should be well rinsed and don’t do after chewing gum)
1% Periodic Acid
- Periodic acid 5g
- Distilled water 500 ml
Store refrigerated
Expiry: 2 months
Schiff’s Reagent
- Basic fuchsin dye (CI 42500) 1g
- 1 N hydrochloric acid* (acidity) 25 ml
- Sodium metabisulphite (source of sulphur) 3.8 g
- Distilled water 75 ml
- Shake vigorously and dissolve overnight
- Add several teaspoons of activated charcoal; shake well 2 min.
- Let stand 5 min.; filter and refrigerate
| * 1N HCL: distilled water | 916.5 mL |
| Concentrated HCL | 83.5 mL |
| Add acid to water carefully. | |
We use purchased. Store in refrigerator.
Harris’ haematoxylin
- Haematoxylin – 5g.
- 100% alcohol – 50mls
- Potassium alum – 100g (mordant)
- Distilled water – 1 litre
- Mercuric oxide – 2.5g (alternatively, sodium iodate may be used as an oxidizer)
- Glacial acetic acid – 40mls
Dissolve the potassium alum in the water by warming and stirring. Dissolve the haematoxylin in the alcohol and add. Bring rapidly to the boil remove from heat and add the mercuric oxide. Cool, add the acetic acid and filter. Ready for use immediately.
We use purchased.
Safety
- Hydrochloric acid is caustic use caution, flush with water. Wear gloves, goggles and lab coat.
- Schiff’s Reagent: Use extreme caution, Basic fuchsin (pararosaniline) is a known carcinogen. Wear gloves, goggles, particle mask and lab coat, while preparing solution. Work under the hood, keep hot, uncapped, solutions under the hood.
- Avoid contact with and inhalation of dyes. Note: we use purchased.
METHOD
- Deparaffinize and bring sections to distilled water. Hold the sections labeled “PAS” in water.
- After rinsing mouth, spit on the slides labeled “PAS/D” and lay horizontally over the top of a large coplin jar for 20 minutes. Do not over digest.
- Rinse well in running tap water then rinse in distilled. “Residual” spit may stain around the tissue if not well rinsed.
- Place all sections in 1% periodic acid for 8 mins
- Wash for 10 minutes, drain well
- Place in Schiff’s reagent in a plastic coplin jar for 15 mins with lid on
- Wash in fast running tap water for 20 mins
- Stain nuclei with haematoxylin 1 min
- Rinse in running tap water
- Blue nuclei in lithium carbonate
- Rinse in running tap water
- Dehydrate, clear and mount.
Results
Nuclei blue
Glycogen will be stained magenta on the PAS stained slide and will be absent on the PAS/D stained slide.
Notes
- If slide is over-digested, the tissue must be re-cut. Over digestion has the appearance of lace; there is no tissue left.
- Store both Periodic Acid and Schiff’s Reagent in fridge. However, solutions should be at room temperature (R.T.) for use. Periodic Acid and Schiff’s Reagent can be discarded down the sink with copious amounts of water.
- Schiff’s Reagent should be clear, colorless with a pungent odor.
- If crystals form, Schiff’s Reagent can be used once filtered.
- Sulfite rinses x 3 may be used optionally after Schiff’s:
| % Potassium metabisulfite | 7.5 mL |
| N/1 HCL | 7.5 mL |
| DH2O | 135.0 mL |
Control
Identical sections (serial) are obtained on two separate slides labeled PAS and PAS/D. One is digested the other is not. Both are stained with the PAS stain. Liver is a good control for PAS/PAS-D, while kidney provides a more sensitive control for the PAS reaction only.
References
IWK. (2007). IWK Laboratory Histology Staining Manual, Special Stains.
Carson, F. L., & Cappellano, C. H. (2015). Histotechnology: A Self-Instructional Text (4th Ed.). American Society of Clinical Pathologists Press.