Introduction
- Carbohydrates play essential roles in biological structures and functions.
- Staining techniques are used to detect different carbohydrate classes, including neutral mucopolysaccharides, acid mucins, and glycogen.
Special Stains for Carbohydrates
These stains help identify glycogen, mucins, and acidic polysaccharides in tissues.
Periodic Acid-Schiff (PAS) Stain
Principle:
- PAS staining detects neutral mucopolysaccharides, glycogen, glycoproteins, and basement membranes.
- Periodic acid oxidizes 1,2-glycol groups in carbohydrates to form aldehydes.
- These aldehydes react with Schiff’s reagent, forming a magenta color.
- PAS stain is widely used in detecting fungal infections, glycogen storage diseases, basement membrane thickening, and adenocarcinomas.
Reagents:
- Periodic Acid (0.5–1%) – Oxidizing agent that converts glycol groups to aldehydes.
- Schiff’s Reagent (Basic Fuchsin + Sulfurous Acid) – Reacts with aldehydes, producing a magenta color.
- Sulfurous Acid or Metabisulfite Rinse – Removes excess Schiff’s reagent to prevent background staining.
- Hematoxylin (counterstain, optional) – Stains nuclei blue for contrast.
Procedure:
- Deparaffinize slides in xylene and rehydrate through graded alcohols.
- Treat with 0.5–1% periodic acid for 5–10 minutes to oxidize carbohydrates.
- Rinse with distilled water.
- Stain with Schiff’s reagent for 15–20 minutes.
- Wash with running tap water for 5–10 minutes (color develops).
- Counterstain with hematoxylin if required.
- Dehydrate, clear, and mount in synthetic resin.
Results:
- Glycogen, basement membranes, mucins, and fungal walls → Magenta
- Nuclei → Blue (if counterstained)
Applications:
- Detection of fungal infections (e.g., Candida, Cryptococcus).
- Identification of glycogen storage diseases.
- Basement membrane thickening in diabetes mellitus.
PAS with Diastase (PAS-D) Stain
Principle:
- PAS-D stain differentiates glycogen from other PAS-positive substances.
- Diastase (amylase enzyme) digests glycogen, removing its PAS staining.
Reagents:
- Same as PAS stain, but includes diastase enzyme.
Procedure:
- Prepare two slides from the same tissue sample.
- Treat one slide with diastase (salivary amylase) at 37°C for 30 minutes to digest glycogen.
- Stain both slides using the PAS protocol.
Results:
- Diastase-treated slide (PAS-D): Glycogen disappears.
- Untreated slide (PAS-only): Glycogen remains PAS-positive (magenta).
Applications:
- Used to confirm the presence of glycogen, particularly in liver and muscle diseases.
Alcian Blue Stain (pH 2.5 and pH 1.0)
Principle:
- Alcian blue selectively stains acid mucopolysaccharides (mucins) based on their charge and pH.
- At pH 2.5, it stains both carboxylated and sulfated mucins.
- At pH 1.0, it stains only sulfated mucins.
Reagents:
- Alcian Blue Solution (pH 2.5 or 1.0)
- Acetic Acid (3%) 100 ml
- Alcian Blue 8GX 1 g
- Nuclear Fast Red
- Aluminum sulfate 5g
Distilled water 100ml
Nuclear fast red 0.1g
- Aluminum sulfate 5g
Dissolve the aluminum sulfate in the water with heat. Add the nuclear fast red to water while still hot and filter.
Procedure:
- Dewax in xylene and rehydrate through graded bethanols to distilled water.
- Stain in the alcian blue solution for 30 minutes.
- Rinse in running tap water for 5 minutes.
- Counterstain in nuclear fast red for 10 minutes.
- Wash in running tap water for 1 minute.
- Dehydrate in graded ethanol.
- Clear in xylene and mount in a miscible medium.
Results:
- Acid mucins → Blue
- Nuclei → Pink/red
Applications:
- Used in adenocarcinomas, Barrett’s esophagus, and mucopolysaccharidoses.
Mucicarmine Stain
Principle:
- Mucicarmine selectively stains epithelial mucins.
Solutions
Southgate’s mucicarmine stock solution
Carmine (alum lake) 1g
Aluminum hydroxide 1g
50% ethanol 100ml
Add the above reagents to a 500 ml Pyrex flask. Shake well and add 0.5 g of anhydrous aluminum chloride. Place the flask in a boiling water bath; bring the solution to a boil. Agitate while boiling for 2.5 to 3 minutes. Cool the flask under running tap water. Filter and store at 4°C. Stable for several months.
Mucicarmine working solution
Southgate’s mucicarmine stock solution 10ml
Distilled water 90ml
Alcoholic hematoxylin
Hematoxylin 1g
Ethanol (95%) 100ml
Acidified ferric chloride stock solution
Ferric chloride 2.48g
Distilled water 97ml
Concentrated hydrochloric acid (HCl) 1ml
Weigert’s iron hematoxylin working solution
Alcoholic hematoxylin 50ml
Acidified ferric chloride solution 50ml
This solution should be mixed just before use.
Metanil yellow working solution
Metanil yellow 0.25g
Distilled water 100ml
Glacial acetic acid 0.25ml
Mix and store in a brown bottle or a bottle completely wrapped with aluminum foil.
Procedure:
- Dewax with xylene and rehydrate through graded ethanols to water.
- Stain in Weigert’s iron hematoxylin working solution for 10 minutes.
- Rinse in running tap water for 10 minutes.
- Stain in the mucicarmine working solution for 30 minutes.
- Rinse slides in two changes of distilled water.
- Stain in the metanil yellow working solution for 30–60 seconds.
- Rinse quickly in distilled water.
- Dehydrate in graded ethanols and clear in xylene.
- Coverslip using a miscible mounting medium
Results:
- Epithelial mucins → Pink/red
- Nuclei → Black
- Background → Yellow
Applications:
- Used for mucin-secreting tumors (e.g., adenocarcinomas).
Combined alcian blue-PAS stain
Fixation
Any fixative.
Sections
Paraffin wax processed or frozen.
Solutions
Alcian blue solution (in 3% acetic acid)
Periodic acid solution
Procedure
1. Dewax in xylene and rehydrate through graded ethanols to distilled water.
2. Stain in the alcian blue solution for 30 minutes.
3. Rinse in running tap water for 5 minutes and then briefly in distilled water.
4. Oxidize with periodic acid for 5 minutes.
5. Rinse in running tap water for 5 minutes.
6. Cover the sections with Schiff reagent for 15 minutes.
7. Rinse in running tap water for 10 minutes.
8. Stain lightly with hematoxylin.
9. Rinse in running tap water for 5–10 minutes and blue in an appropriate blueing solution.
10. Rinse in tap water for 5 minutes.
11. Dehydrate in graded ethanols, clear with xylene, and mount with a miscible medium.
Results
Glycogen, neutral mucins, various glycoproteins – Magenta
Acid mucins (sulfomucins and sialomucins) – Blue
Proteoglycans and hyaluronic acid – Blue
Colloidal iron stain
Solutions
Stock colloidal iron solution
Bring 250 ml of distilled water to a boil and add 4.4 ml of a 29% ferric chloride solution (USP XI). Continue to boil until the solution turns dark red, at which time the solution should be removed from the heat and allowed to cool. This solution is stable for one year.
Colloidal iron working solution
Stock colloidal iron solution 20ml
Distilled water 15ml
Glacial acetic acid 5ml
Prepare just prior to use.
Acetic acid (12%) solution
Glacial acetic acid 24ml
Distilled water to make up to 200 ml
Potassium ferrocyanide (5%) solution
Potassium ferrocyanide 5 g
Distilled water 100 ml
Hydrochloric acid (5%) solution
Concentrated hydrochloric acid 5ml
Distilled water 95ml
Potassium ferrocyanide-hydrochloric acid
5% potassium ferrocyanide solution 50ml
5% hydrochloric acid solution 50ml
Mix just prior to use.
Acid fuchsin stock (1%)
Acid fuchsin 1g
Distilled water 100ml
van Gieson working solution
1% acid fuchsin stock 5ml
Saturated picric acid 95ml
Procedure
1. Dewax in xylene and rehydrate through graded ethanols to water.
2. Rinse in 12% acetic acid solution for 1 minute.
3. Cover the sections with the colloidal iron working solution for 1 hour.
4. Rinse in four changes of the 12% acetic acid solution (3 minutes each).
5. Place in the potassium ferrocyanide-hydrochloric acid solution for 20 minutes.
6. Rinse in running tap water for 5 minutes.
7. Rinse briefly in distilled water.
8. Stain with van Gieson working solution for 5 minutes.
9. Dehydrate the specimens in 95% ethanol and absolute ethanol, three changes each. Clear in xylene.
10. Coverslip using an appropriate mounting medium.
Results
Proteoglycans, hyaluronic acid and acidic mucins – bright blue
Collagen – Red
Muscle and cytoplasm – Yellow
Azure A stain
Alcoholic azure A solution
Azure A 0.01g
Ethanol 30% 100ml
Procedure
1. Dewax sections in xylene and hydrate through graded ethanols to distilled water.
2. Cover sections with the azure A solution for 10 minutes.
3. Rinse in distilled water.
4. Dehydrate the sections in graded ethanols and clear in xylene.
5. Coverslip using a miscible mounting medium.
Results
Acid mucins and proteoglycans – purple to red
Tissue background – blue