PAS with Diastase: Staining Protocol
Sample Type Required: Deparaffinized and re-hydrated tissue sections on positively charged slides.
Fixative: 10% Neutral Buffered Formalin
| Step | Reagent | Time | Technical Notes |
| 1 | Malt Diastase Solution | 1 hour at 37C | Solution should be preheated to 37C. Do not heat the solution above 40C as enzyme activity can destroyed at higher temperatures. Insufficient heat may cause incomplete digestion of glycogen. |
| 2 | Distilled Water | 3 Changes | Rinse slides gently to remove diastase. |
| 3 | Periodic Acid 0.5% Solution | 5 Minutes |
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| 4 | Distilled Water | 3 Changes |
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| 5 | Schiff Reagent | 15 Minutes | Gluteraldehyde should be avoided as a tissue fixative since it is a dialdehyde that will react with this reagent and give false-positive PAS staining. |
| 6 | Potassium Metabisulfite 0.55% Solution | 1 Minute in 2 Changes | While some technicians omit this reagent step in the protocol with no problems, metabisulfite rinses may be necessary to remove any excess leucofuchsin left over after exposure to the Schiff reagent. Highly chlorinated water can cause these excess molecules to nonspecifically stain other tissue elements in the section. |
| 7 | Running Tap Water | Up to 10 Minutes | Warm to hot running water develops the PAS stain and gives a more intense staining reaction than cold water. |
Expected Results - Glycogen will be absent from tissue section
The following elements will show positive PAS (Rose Red) staining
- Neutral Mucins
- Some Epithelial Mucins
- Basement Membranes
- Fungal Walls
Post Staining Procedure: Tissue sections should be rinsed well in distilled water, dehydrated with 95% and absolute alcohols, cleared and cover-slipped.
