Periodic Acid-Schiff (PAS) with Diastase: Staining Protocol

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Periodic Acid-Schiff (PAS) with Diastase: Staining Protocol

Sample type required: Deparaffinized and re-hydrated tissue sections on positively charged slides.

Fixative: 10% NBF
StepReagentTimeTechnical Notes
1 Malt diastase solution 1 hour at 37° C Solution should be preheated to 37° C.
Do not heat the solution above 40° C, as enzyme activity can destroyed at higher temperatures.
Insufficient heat may cause incomplete digestion of glycogen.
2 Distilled water3 changesRinse slides gently to remove diastase.
3 Periodic acid:
0.5% solution
5 minutes
4 Distilled water 3 changes
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 must be counterstained to fully recognize the presence of PAS positive staining. This can be achieved with the use of hematoxylin (see separate page for specific guidelines).
  • After counterstain, tissue sections should be rinsed well in distilled water, dehydrated with 95% alcohol, followed by 100% alcohols, cleared with xylene (or xylene substitute), and cover-slipped.

PAS with diastase; liver