Periodic acid-Schiff (PAS) is another commonly used special stain in the histology lab.
What Does PAS Stain?
(1) Polysaccharides: The technique is commonly used to identify polysaccharides- these macromolecules are composed of monosaccharide units joined by covalent bonds. The main polysaccharide identified via histology staining in human and animal tissue sections is glycogen. This is present in numerous tissues, including skeletal muscle, cardiac muscle, liver, and kidney.
(2) Neutral mucus substances: It is also commonly used to stain and identify neutral mucus substances. These can include glycoproteins, glycolipids, and neutral mucins, which are produced by epithelial cells in different organs.
(3) Tissue basement membranes: These PAS-positive thin layers of reticular connective tissue anchor and support epithelium and endothelium to underlying connective tissue.
(4) Fungal organisms: The cell walls of some fungal organisms contain high levels of carbohydrate, and also stain PAS-positive. However, this only works on living fungi.
General Principles of the Stain
The reactivity of the PAS technique is based on the structure of the monosaccharide units.
The first reaction in the stain involves periodic acid acting as an oxidising agent to oxidise the carbon-to-carbon bonds between two adjacent hydroxyl groups. This produces Schiff reactive aldehyde groups.
In the second reaction, the tissue section reacts with Schiff reagent. This comprises a mixture of basic fuchsin, hydrochloric acid, and sodium metabisulphite. The basic fuchsin in the mixture reacts with newly formed aldehyde groups in the tissue to produce a bright magenta colour.
Finally, when the section is rinsed in water, bound fuchsin molecules in the tissue then produce a bright magenta colour. The intensity of the colour is proportional to the concentration of hydroxyl groups originally present in the monosaccharide units.
Haematoxylin is then typically used as a counter stain to visualise other tissue elements. When PAS is used to demonstrate fungal organisms, however, a light green counter stain is preferred.
Diastase (alpha-amylase) digestion may also be used to assist in the diagnosis of glycogen storage diseases. Diastase hydrolyses starch, glycogen, and breakdown products originating in these tissue polysaccharides. When compared to a slide of tissue containing glycogen, a diastase extraction slide will have no visible PAS stain.
|Histologic feature||Colour with PAS Stain|
|Glycogen, basement membranes, mucus substances, fungal organisms||Magenta|
|Nuclei||Blue (with haematoxylin counter stain)|
|Other tissue elements||Green (with light green counter stain)|
Who Uses PAS Stain?
This stain is used widely for both diagnostic and research purposes. For example, researchers studying glycogen storage diseases or diseases of the basement membrane may routinely examine PAS-stained tissues sections to evaluate these respective elements in tissues of interest.
In diagnostic labs, pathologists often use this stain to help answer questions that may arise after examining routine haematoxylin and eosin-stained sections. In particular, it can be used to help with the diagnosis of:
- Glycogen storage diseases: These are conditions in which excessive quantities of glycogen are stored in the liver, muscles, or kidney. PAS is often routinely used in the clinic to demonstrate glycogen accumulation in biopsies of these tissues.
- Tumors: Glycogen granules can also be present in some tumors, including some of those that arise in tissues such as the pancreas, lung, and bladder.
- Fungal infection: PAS can be used to visualise some fungal organisms in tissue sections.
- Basement membranes: Since PAS can be used to highlight the basement membranes of tissues, it can be used to identify disorders in which there is weakness or improper functioning of basement membranes – such as in the case of some glomerular diseases in the kidney.
What is PAS most commonly used for in your lab?