Principle of PAS Staining
PAS method works by exposing the tissue to periodic acid. Periodic acid acts as oxidizing agent which oxidizes compounds having free hydroxyl group (-OH group) or amino/alkylamine group resulting in dialdehydes. These dialdehydes when exposed to Schiff’s reagent, an insoluble magenta colored complex is formed. A suitable basic stain is used as counter stain.
Preparation of staining solutions
1) Periodic Acid Solution :
- Periodic Acid : 1 gram
- Distilled water : 100 ml
2) Schiff’s Reagent :
- Fuchsin Basic : 1 gm
- Distilled water : 100 ml
- Sodium metabisulphite : 2 gm
- Conc. HCl : 2 ml
- Charcoal activated : 0.3 gm
Dissolve basic fuchsin in boiling water, cool at 50°C and filter. Add sodium metabisulphite and HCl. Store at dark room at room temperature overnight. Add charcoal, shake for one minute and filter
Procedure of PAS Staining
- Bring sections to distilled water.
- Treat with periodic acid for 5 minutes.
- Rinse well in distilled water.
- Cover with Schiff’s reagent for 5-15 minutes.
- Wash in running tap water for 5-10 minutes
- Counter stain with Herri’s hematoxylin for approximately 15 seconds.
- Differentiate (if necessary) with acid alcohol and bluing as usual.
- Wash in tap water.
- Rinse in increasing concentration of alcohol (70, 80, 95 and 100%)
- Clear in xylene and mount as usual.
Formation of insoluble magenta colored complex denotes positive result.
Uses of PAS Staining
PAS stain is mainly used to highlight the molecules (structures) with high percentage of carbohydrate content such as glycogen, glycoproteins, and proteoglycans typically found in connective tissue, glycocalyx and basal laminae.
PAS staining can be used to assist in the diagnosis of several medical conditions such as:
- Glycogen storage disease (vs. other storage disease)
- Adenocarcinoma which often secretes mucin
- Paget’s disease of breast
- Alveolar soft part sarcoma
- Staining macrophages in Whipple’s disease
- Erythroleukemia, Leukemia of immature RBCs
- Fungal infection (cell wall stain magenta)
Differentiation is the process of removing excess dyes from tissues. It is similar to decolorizing, but infers with high degree of selectivity. Differentiation can be accomplished by: Solvents (e.g. tap water in H&E staining), pH control, Mordants, Oxidizers, Other Dyes
Bluing step converts the initial soluble red color (of hematoxylin) within the nucleus to an insoluble blue color. Some examples of bluing solution (alkaline pH) are: ammonia water, dilute lithium carbonate, Scott’s tap water (potassium carbonate, magnesium sulphate and water
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