Periodic Acid-Schiff Stain

Table of contents

Periodic Acid-Schiff (PAS) stain is a widely used histochemical technique highlighting glycogen, mucopolysaccharides, glycoproteins, and other carbohydrate-rich tissue structures.
It is commonly employed in pathology for diagnosing fungal infections, glycogen storage diseases, basement membrane abnormalities, and certain types of tumors.
The PAS stain is particularly useful in identifying carbohydrate-laden components of cells and extracellular matrices, aiding in disease identification and classification.

PAS staining is based on the oxidation of carbohydrates by periodic acid, which generates aldehyde groups.
These aldehydes react with Schiff’s reagent to produce a magenta-colored complex, allowing visualization of carbohydrate-containing structures.
The reaction is highly specific to polysaccharides, mucopolysaccharides, glycoproteins, and glycolipids, making PAS a valuable histopathological tool.
The intensity of PAS staining depends on the concentration and distribution of carbohydrates within the tissue.

Microscope slides with properly fixed and sectioned tissue samples (paraffin-embedded or frozen sections)
Staining rack for slide handling
Glass beakers and cylinders for reagent preparation and application
Distilled water for rinsing steps
Timer to ensure precise incubation times
Coverslips and mounting medium for slide preservation
Light microscope for examination

Periodic Acid Solution (0.5-1%) – Oxidizes carbohydrates to aldehydes, making them reactive to Schiff’s reagent.
Schiff’s Reagent – A colorless solution that reacts with aldehydes to form a magenta-colored complex upon binding.
Sulfurous Acid or Sodium Metabisulfite Solution – Used to remove excess Schiff’s reagent, preventing non-specific staining.
Hematoxylin (optional counterstain) – Stains nuclei blue, providing contrast for better visualization.
Ethanol and Xylene – Used for dehydration, clearing, and slide preparation for microscopic evaluation.
Diastase Enzyme (for PAS-D) – Used in diastase-PAS staining to digest glycogen, differentiating glycogen from other PAS-positive substances.

Deparaffinization and Hydration:
- Place tissue sections in xylene for 5-10 minutes to remove paraffin.
- Rehydrate slides through graded ethanol solutions (100%, 95%, 70%) down to distilled water.
Oxidation:
- Immerse slides in 0.5-1% periodic acid solution for 5-10 minutes.
- Rinse well in distilled water to remove excess periodic acid.
Schiff’s Reagent Reaction:
- At room temperature, Incubate slides in Schiff’s reagent for 10-15 minutes.
- Observe the tissue sections turning a faint pink color.
- Rinse slides in sulfurous acid or sodium metabisulfite solution (3 changes, 1-2 minutes each) to remove excess stain.
- Wash slides in running tap water for at least 5 minutes until the pink color intensifies.
Counterstaining (if necessary):
- Stain slides with hematoxylin for 1-2 minutes to highlight nuclei.
- Rinse with tap water and blue in an alkaline solution if required.
Dehydration and Mounting:
- Dehydrate slides through graded ethanol solutions (70%, 95%, 100%).
- Clear in xylene and mount with a permanent mounting medium.
Microscopic Examination:
- Examine under a light microscope at appropriate magnifications.

PAS-positive structures: Magenta/purple (glycogen, mucins, basement membranes, fungal cell walls, some types of tumor cells)
PAS-negative structures: Unstained or background counterstained with hematoxylin (blue nuclei)
PAS-D (Diastase PAS) results:
- Glycogen-positive areas disappear after diastase treatment (confirming glycogen content).
- Other PAS-positive components remain unchanged (e.g., mucins, fungal walls, basement membranes).

Identification of glycogen storage diseases (PAS-positive, diastase-sensitive glycogen deposits).
Diagnosis of fungal infections (fungal cell walls stain magenta with PAS).
Detection of mucin-secreting adenocarcinomas.
Examination of renal and hepatic diseases involving basement membrane changes.
Analysis of muscle and connective tissue disorders involving glycoproteins.

Highly sensitive for detecting glycogen, mucins, and polysaccharides.
Useful in diagnosing fungal infections, basement membrane abnormalities, and certain tumors.
Provides good contrast when combined with hematoxylin counterstaining.
It can be used alongside diastase digestion (PAS-D) to confirm glycogen presence.

Non-specific staining can occur if not properly controlled.
Cannot distinguish between different types of carbohydrates without additional techniques.
Schiff’s reagent is light-sensitive and must be stored carefully to maintain its efficacy.
Requires well-fixed tissue samples, as improper fixation can affect staining quality.

Does not differentiate between various types of polysaccharides and glycoproteins.
It may produce false positives due to contaminants or improper fixation.
Additional confirmatory tests (e.g., diastase digestion) are required to identify glycogen specifically.
Staining results may vary based on fixation and tissue processing techniques.