Alcian blue with periodic acid-Schiff (PAS) stain

The Alcian blue with periodic acid-Schiff (PAS) stain is a combination staining technique used in histology to detect and differentiate acidic and neutral mucins, as well as other carbohydrates, in tissue sections. This dual staining method allows for the identification and localization of both acidic and neutral mucosubstances in various tissues.

Principle:

The principle of the Alcian blue with PAS stain involves two different staining reactions:

• Alcian blue staining: Alcian blue dye binds to acidic mucins, highlighting their presence. The dye forms a complex with the acid mucopolysaccharides and glycosaminoglycans (GAGs) present in the tissue sections.
• Periodic acid-Schiff (PAS) staining: The PAS reaction involves the oxidation of carbohydrates present in the tissue sections by periodic acid, followed by the reaction of the oxidized carbohydrates with Schiff's reagent. This reaction results in the formation of a pink or magenta color, indicating the presence of neutral mucins and other carbohydrates.

Requirements:

To perform an Alcian blue with PAS stain, the following requirements are necessary:

• Prepared tissue sections: Thin tissue sections fixed with a suitable fixative, such as formalin, and embedded in paraffin.
• Alcian blue solution: A working solution of Alcian blue dye at an appropriate pH, typically 1.0 or 2.5.
• Periodic acid solution: A freshly prepared 1% periodic acid solution for the oxidation step.
• Schiff's reagent: A commercially available or in-house prepared Schiff's reagent, used to react with the oxidized carbohydrates.
• Nuclear counterstain: Harris hematoxylin or any suitable nuclear stain for counterstaining cell nuclei.
• Distilled water: Required for diluting and rinsing staining solutions.
• Laboratory equipment: This includes glass slides, coverslips, staining racks, staining dishes, and a microscope for examination.

Procedure:

The procedure for performing the Alcian blue with PAS stain involves the following steps:

1. Deparaffinization and hydration: Remove paraffin from the tissue sections using xylene or a xylene substitute, followed by rehydration through graded alcohols.
2. Alcian blue staining: Immerse the tissue sections in an Alcian blue solution (pH 1.0 or 2.5) for a specified time, usually ranging from 15 minutes to overnight.
3. Rinse: Rinse the tissue sections with distilled water to remove excess Alcian blue dye.
4. Oxidation: Incubate the tissue sections in a freshly prepared 1% periodic acid solution for 5-10 minutes to oxidize the carbohydrates.
5. Rinse: Rinse the tissue sections with distilled water to remove excess periodic acid.
6. Schiff's reagent: Add Schiff's reagent to the tissue sections and incubate for 15-30 minutes. This step reacts with the oxidized carbohydrates, producing a pink or magenta color.
7. Counterstaining: Perform nuclear counterstaining with Harris hematoxylin or a suitable nuclear stain for a short period.
8. Dehydration: Dehydrate the tissue sections with graded alcohols.
9. Clearing: Clear the tissue sections with xylene or a xylene substitute.
10. Mounting: Apply a mounting medium and cover the slides with coverslips.

Results:

After staining with Alcian blue with PAS, the following results can be observed:

• Acidic mucins and GAGs: These substances appear blue due to the Alcian blue staining.
• Neutral mucins and carbohydrates: These substances appear pink or magenta due to the PAS reaction.
• Cell nuclei (if counterstained): Nuclei may be stained with a contrasting color, such as blue or purple.

Quality Control (QC):

To ensure accurate staining results, the following QC measures can be taken:

• Use appropriate positive and negative controls to validate staining results. Positive controls can be tissues known to contain acidic and neutral mucins, while negative controls can be tissues that do not have these substances.
• Maintain consistency in staining protocols, including timing, pH of the Alcian blue solution, and concentration of periodic acid.

Interpretations:

Interpretation of the Alcian blue with PAS stain involves evaluating the presence, distribution, and intensity of acidic and neutral mucins in the tissue sections. The blue staining indicates the presence of acidic mucins and GAGs, while the pink or magenta staining indicates the presence of neutral mucins and carbohydrates. The pattern and intensity of staining can provide valuable information about the composition and distribution of mucins and carbohydrates in different tissues.