PAS Staining (Periodic Acid–Schiff, PAS) Standard Operating Procedure (SOP)

I. Purpose and Principle

1.1 Scope of application

This SOP is applicable to:

(1) Demonstration of polysaccharide-related substances (glycogen, neutral mucopolysaccharides, glycoproteins, basement membrane components, etc.) in paraffin-embedded tissue sections and frozen sections.

(2) Semi-quantitative observation of cytoplasmic glycogen and glycoprotein granules in peripheral blood smears and bone marrow smears.

(3) Visualization of PAS-positive structures such as the glomerular basement membrane, neutral mucin in colonic goblet cells, fungi, and amoebic trophozoites.

1.2 Staining principle

(1) Periodic acid (HIO₄) oxidizes vicinal glycol groups on adjacent carbon atoms within polysaccharides to generate aldehyde groups (–CHO).

(2) The aldehyde groups react with the colorless fuchsin derivative in Schiff reagent via a condensation reaction, restoring the magenta chromophore and producing magenta to red deposits.

(3) After nuclear counterstaining with hematoxylin, a clear contrast is formed: “PAS-positive structures in red/magenta and nuclei in blue,” facilitating interpretation.

II. Reagents and Materials

2.1 Specimens and fixation

(1) Tissue specimens

① Fixation: commonly fixed in 10% neutral buffered formalin; if glycogen demonstration is the primary goal, Carnoy’s fixative may be used:

Absolute ethanol 60 mL

Glacial acetic acid 10 mL

Chloroform 30 mL

② Paraffin embedding and sectioning (typical thickness 3–5 μm).

(2) Blood smears / bone marrow smears

① Prepare fresh smears and air-dry.

② Fix in absolute methanol for 5–10 min.

2.2 Main reagents

(1) Periodic acid working solution

For tissue sections:

① 0.5%–1.0% aqueous periodic acid solution.

② Prepare fresh before use or store short-term (within 2 weeks) at 2–8°C protected from light.

Reference formulation (alcohol-based for tissue):

Periodic acid (HIO₄·2H₂O) 0.4 g

95% ethanol 35 mL

0.2 mol/L sodium acetate solution 5 mL

Distilled water 10 mL

(2) Schiff reagent (Schiff solution)

Example basic formulation:

① Dissolve basic fuchsin 0.5 g in 100 mL distilled water; heat to gentle boiling with agitation until fully dissolved.

② Cool to ~50°C, filter, then add 10–20 mL of 1 mol/L hydrochloric acid (HCl) and mix well.

③ When cooled to 25°C, add sodium metabisulfite 0.5–1.0 g and mix thoroughly.

④ Store in an amber bottle at room temperature, protected from light for at least 24 h until the solution becomes nearly colorless.

⑤ If the solution remains noticeably red, add 1–2 g activated charcoal for adsorption and filter.

⑥ Store at 2–8°C protected from light; do not use once the solution turns red.

(3) Hematoxylin solution

Harris or Mayer hematoxylin may be used; alternatively, the following formulation may be prepared:

① Hematoxylin 0.9 g dissolved in 10 mL of 95% ethanol.

② Alum (ammonium alum or potassium alum) 20 g dissolved in 200 mL distilled water (heat to assist dissolution if needed).

③ Slowly add the alcoholic hematoxylin solution to the alum solution, mix, and heat to boiling.

④ Add mercuric oxide 0.5 g and stir rapidly; when the solution becomes deep purple, immediately cool in cold water.

⑤ Stand overnight, filter; before use, add 5 mL glacial acetic acid to 95 mL hematoxylin solution.

⑥ Store at room temperature protected from light.

(4) Sulfite rinse (optional; for reducing background after tissue PAS staining)

1% sodium metabisulfite 10 mL

1 mol/L hydrochloric acid (HCl) 10 mL

Distilled water 180 mL

(5) Other routine reagents

① Xylene (for deparaffinization and clearing).

② Graded ethanol series (100%, 95%, 80%, 70%, etc.).

③ Tap water / distilled water.

④ Neutral mounting medium (neutral resin).

2.3 Major equipment

(1) Microtome, water bath for floating sections, slide oven.

(2) Microscope.

(3) Incubator (optional).

(4) Staining jars, slides, coverslips, etc.

III. Procedure

3.1 PAS staining for paraffin tissue sections

(1) Deparaffinization and rehydration to water

① Bake paraffin sections at 60°C for 30–60 min.

② Xylene, 2 changes, 10 min each.

③ 100% ethanol, 2 changes, 5 min each.

④ 95% ethanol, 80% ethanol, 70% ethanol, 2–3 min each.

⑤ Rinse with tap water to bring sections into water.

(2) Oxidation (periodic acid treatment)

① Incubate sections in 0.5%–1.0% periodic acid solution for 5–10 min (commonly 10 min).

② Rinse thoroughly in tap water for 5–10 min to remove residual oxidant.

(3) Schiff reagent staining

① Incubate sections in Schiff reagent for 10–20 min (commonly 10–15 min).

② Rinse under running tap water for 5–10 min until the background clears and becomes faintly pink.

③ If background remains strong, extend running-water rinse or briefly treat with sulfite rinse followed by running-water rinse.

(4) Nuclear counterstaining with hematoxylin

① Stain in hematoxylin for 2–5 min (adjust according to stain strength).

② Rinse in tap water for 5 min.

③ If nuclei are overstained, differentiate briefly in 1% acid alcohol for a few seconds, then “blue” in tap water for several minutes.

(5) Dehydration, clearing, and mounting

① Dehydrate through graded ethanol (70% → 95% → 100%), 1–2 min each.

② Clear in xylene, 2 changes, 5 min each.

③ Mount with neutral resin mounting medium and apply coverslip, avoiding air bubbles.

3.2 PAS staining for blood smears / bone marrow smears

(1) Fixation

① Air-dry fresh blood or bone marrow smears.

② Fix in absolute methanol for 5–10 min, then air-dry.

(2) Oxidation

① Incubate in 10 g/L (1%) aqueous periodic acid solution for 20 min.

② Rinse several times with distilled water; gently flick off excess liquid or air-dry.

(3) Schiff reagent staining

① Cover the smear with Schiff reagent or incubate in a staining jar for 45–60 min (commonly 60 min).

② Rinse gently under running tap water for 10–15 min to remove free dye and lighten background.

(4) Hematoxylin staining

① Stain in hematoxylin for 5–10 min.

② Rinse in tap water for 10–15 min and air-dry.

(5) Smears may be examined directly under oil immersion, or mounted as needed.

IV. Result Interpretation

4.1 Tissue sections

(1) PAS-positive structures

① Glomerular basement membrane and vascular basement membrane: uniform red or magenta thickened bands.

② Colonic goblet cells and mucous gland secretions: neutral mucin stains red.

③ Glycogen in hepatocytes and skeletal muscle: cytoplasmic granules or diffuse red staining.

④ Fungal hyphae/spores and amoebic trophozoite cell walls: distinct red staining.

(2) Nuclei

Blue or blue-purple after hematoxylin counterstaining.

4.2 Blood smears / bone marrow smears

(1) Positive reaction

① Platelets: cytoplasm deep red.

② Neutrophils: cytoplasm red to deep red with PAS-positive granules.

③ Monocytes: cytoplasm pale red, with fine or coarse positive granules.

④ Some lymphocytes: a small number of pale red or red cytoplasmic granules.

⑤ Megakaryocytes: diffuse red or deep red cytoplasm.

(2) Negative or weakly positive

① Normal erythrocytes and nucleated erythroid precursors in bone marrow: typically unstained or negative.

② PAS patterns in certain undifferentiated cells depend on the pathological type and should be interpreted together with morphology and other stains.

V. Quality Control and Notes

5.1 QC key points

(1) Include known PAS-positive tissue (e.g., kidney, colon) as a positive control in each batch.

(2) Verify periodic acid and Schiff reagent performance:

① Periodic acid solution should be clear and free of precipitate; replace if beyond the recommended storage period.

② Schiff reagent should be colorless or very pale; obvious reddening indicates oxidation and loss of activity.

(3) Insufficient running-water rinsing commonly leads to high background; overly long rinsing generally does not eliminate positive structures but may reduce contrast.

5.2 Common issues and troubleshooting

(1) Overall staining too weak

① Periodic acid concentration too low or oxidation time insufficient.

② Schiff reagent inactivated or incubation time insufficient.

③ Verify preparation and storage conditions.

(2) High background or nonspecific staining

① Running-water rinse time insufficient or sulfite rinse inadequate.

② Improper fixation or sections too thick.

③ Extend running-water rinse or optimize fixation.

(3) Poor cellular morphology in blood smears

① Inappropriate fixation time or insufficient drying.

② Overstaining resulting in unclear nuclear–cytoplasmic boundaries.

③ Adjust fixation time and hematoxylin staining time accordingly.

VI. Safety and Waste Disposal

(1) Periodic acid is an oxidizer. Wear protective gloves and safety goggles during preparation and use; avoid direct contact with organic combustibles.

(2) Schiff reagent involves acid and metabisulfite; ensure adequate ventilation to avoid inhalation of irritating vapors.

(3) Waste liquids containing organic dyes and heavy metals (e.g., mercuric oxide) must be collected and disposed of as chemical hazardous waste; do not discharge into the sewage system.

(4) Follow laboratory biosafety and chemical management regulations throughout the procedure.

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