PAS Reaction
for 1-2 Glycols

Likely the most common application for Schiff's reagent is the Periodic Acid Schiff, or PAS, reaction. This is a technique for the demonstration of carbohydrates in tissue sections. The purpose of the periodic acid is to oxidise some of the tissue carbohydrates. This produces aldehyde groups, which can then condense with Schiff's reagent forming a bright red colouration and demonstrating the tissue component to which the carbohydrate is attached.

The PAS reaction is quite straightforward. Sections are first brought to water, then a 1% aqueous solution of periodic acid is applied for 10 to 30 minutes. The sections are washed to remove any traces of the periodic acid, then the Schiff's reagent is applied for a while, again for 10 to 30 minutes. The Schiff's reagent is rinsed off with water, then the sections are washed with water until the water is clear and the sections look pink, usually about 10 to 30 minutes. The colour deepens somewhat as the washing continues, within limits. The time for which the reagents are applied can affect the intensity of the colour. Also, some substances are known to require extended oxidation, and may not be oxidised with the times given.

The treatment with periodic acid oxidises some of the carbohydrates in the tissue to aldehydes. The carbohydrates involved are 1-2 glycols, shown below in red. Since not all carbohydrates include this structure, the PAS is not a method for carbohydrates in general but only for those which contain 1-2 glycols or closely related structures, such as one in which an amino group replaces one of the hydroxyl groups, also shown below in red. Carbohydrates which stain with this method include polysaccharides, mucopolysaccharides, glycoproteins and glycolipids. The essential point is that treatment must be able to produce an aldehyde on the carbohydrate component.

 

According to Culling, Hotchkiss stated that the following criteria were necessary for a tissue substance to give a positive PAS reaction:–

1. It must contain the 1-2 glycol grouping, an equivalent amino or alkylamino grouping, or the oxidation product CHOH-CO.
2. It must not diffuse away during fixation.
3. It must give an oxidation product which does not diffuse away.
4. It must be present in sufficient concentration to produce a detectable final colour.

The following list, also based on Culling, gives the PAS positive materials usually encountered, although it is not exhaustive:–

• Actinomyces clubs.
• Basement membranes.
• Cartilage.
• Cerebrosides.
• Collagen from areolar connective tissue.
• Compound lipids.
• Glycogen.
• Kerasin (Gaucher’s disease).
• Kidney tubules.
• Megakaryocyte granules.
• Ocular lens capsule.
• Paneth cell granules (some species).
• Pituitary beta cells.
• Retinal rods.
• Starch.

• Mucins of:
  • intestinal tract,
  • peptic gland,
  • uterine cervical glands,
  • salivary glands,
  • conjunctiva,
  • bronchial glands,
  • ovarian follicles and cysts,
  • prostatic gland secretion,
  • corpora amylacea,
• Pancreatic zymogen granules.
• Phospholipids.
• Renal hyaline casts.
• Russell bodies.
• Thyroid colloid.

Other oxidising agents
Periodic acid is not the only oxidising agent that has been recommended for the oxidation of carbohydrates in this kind of reaction, although it is certainly the most used and arguably the most effective. Chromic acid (chromium trioxide in water) is often used, and forms the basis of a method for fungi. It is different from periodic acid in that it continues to oxidise the aldehydes it produces. Over oxidation will therefore eventually lead to a pale or false negative reaction. Some other oxidising agents have been used as well including permanganic acid, performic acid, peracetic acid and lead tetracetate. Although valuable for specific purposes, these are not in common use.

 

Reference
Kiernan. J.A., (1999)
Histological and histochemical methods: Theory and practice, Ed. 3
Butterworth Heinemann, Oxford, UK.

Culling C.F.A., (1963)
Handbook of histopathological techniques Ed. 2
Butterworth, London, UK.

Culling C.F.A., (1974)
Handbook of histopathological and histochemical techniques Ed. 3
Butterworth, London, UK.

Pearse, A. G. E., (1968, 1972)
Histochemistry: Theoretical and Applied, Ed. 3
Churchill Livingstone, Edinburgh, London, UK

Translate in Google Translate Instructions