AB—PAS Staining Kit 

The principle of Alcian Blue staining is based on the interaction between positively charged dye molecules and negatively charged acidic groups in tissues under acidic conditions. When the staining system is adjusted to pH 2.5, the carboxyl groups (—COO^-) in tissues become ionized and form ionic bonds with the cationic Alcian Blue dye. As a result, acidic mucopolysaccharides and acidic mucins containing carboxyl groups (such as hyaluronic acid and epithelial acidic mucins) are stained blue, whereas neutral mucins generally do not react with Alcian Blue and therefore remain unstained.

The principle of Periodic Acid–Schiff (PAS) staining relies on the strong oxidizing property of periodic acid, which specifically oxidizes 1,2-glycol groups (vicinal diols) in carbohydrate molecules to generate aldehyde groups. These aldehyde groups subsequently react with Schiff reagent to form a stable magenta (reddish-purple) compound, producing a characteristic red-purple staining signal under the microscope. This reaction enables clear visualization of glycogen and other polysaccharide-rich or glycoprotein-containing structures in tissues.

MCE AB—PAS Staining Kit combines Alcian Blue staining and PAS staining, allowing the simultaneous detection and differentiation of different types of mucins within the same tissue section. In this method, acidic mucins are typically stained blue, neutral mucins appear red or magenta, and mucins containing both acidic and neutral components may appear purple or bluish-purple. This technique is widely used in histological and pathological studies for the identification and distribution analysis of mucin types within tissues.

Alcian Blue Staining Solution, Periodic Acid Solution, Schiff Reagent: 4°C, 1 year. Protected from light.

Hematoxylin Staining Solution, Acidic Ethanol Differentiation Solution, Bluing Solution: RT, 1 year.

Reagents Preparation

1. Fixative: 10% formalin

2. Other reagents: xylene, neutral mounting medium or mounting reagent, graded ethanol solutions, distilled water, etc.

Procedure

Reagents Preparation

1. Deparaffinization and Rehydration: For paraffin sections, deparaffinize sequentially with xylene, then rehydrate through a graded ethanol series, and finally immerse in distilled water.

2. Alcian Blue Staining: Incubate the sections in Alcian Blue staining solution at room temperature for 10–20 min.

3. Washing: Rinse the sections with distilled water three times, 1–2 min each.

4. Periodic Acid Oxidation: Immerse sections in periodic acid solution and incubate at room temperature for 5-8 min.

5. Schiff Reagent Staining: Incubate sections in Schiff reagent at room temperature in the dark for 10-20 min.

6. Washing: Rinse the sections with running tap water for approximately 10 min.

7. (Optional) Hematoxylin Counterstaining: Counterstain the sections in hematoxylin staining solution for 1–2 min.

8. Differentiation: Differentiate the sections in Acidic Ethanol Differentiation Solution for 2–5 s, followed by rinsing with water.

9. Bluing: Place the sections in bluing solution to allow nuclear bluing, then rinse with water for about 3 min.

10. Dehydration, Clearing, and Mounting: Dehydrate sections through a graded ethanol series, clear with xylene, and mount with neutral resin or mounting medium. Observe results under a microscope.

Interpretation of Results

1. Glycogen, neutral mucins, and glycoproteins: magenta to purplish-red.

2. Acidic mucins: blue.

3. Proteoglycans and hyaluronic acid: blue.

Note: Cells or tissues containing both neutral and acidic mucins may appear varying shades from blue-purple to purple, depending on their relative composition.

1. Since ethanol is highly volatile, graded ethanol solutions should be freshly prepared before use to ensure consistent dehydration performance.

2. Deparaffinization of sections should be thorough; incomplete deparaffinization may compromise staining quality.

3. The oxidation time with periodic acid should not be excessive; the optimal oxidation temperature is 18-22°C.

4. Periodic acid solution and Schiff reagent should be stored at 4°C in a tightly sealed container. Avoid prolonged exposure to light and air during use. Prior to use, it is recommended to equilibrate the reagents to room temperature for 30 minutes while keeping them protected from light.

5. Acidic ethanol differentiation solution should be frequently refreshed. Differentiation time should be adjusted based on section thickness, tissue type, and the age of the differentiation solution. Sufficient rinsing with tap water is required after differentiation.

6. The incubation times in periodic acid and Schiff reagent are critical and should be optimized according to section thickness and tissue type.

7. When counterstaining cell nuclei with hematoxylin, light staining is recommended to avoid obscuring the observation of AB-positive substances. This precaution helps prevent excessive staining of the cytoplasm or mucins, which may mask the blue coloration produced by Alcian Blue.

8. The staining sequence in the AB–PAS combined technique can influence the final staining results. When the PAS staining step is performed before Alcian Blue staining, neutral mucins and glycogen may appear purple. In contrast, when Alcian Blue staining is carried out prior to the PAS procedure, these substances typically exhibit the expected magenta or purplish-red coloration. Therefore, careful control of the staining order is important to obtain optimal staining results.

9. Once opened, reagents should be used promptly to ensure consistent staining performance i