H&E Staining SOP

1. Purpose

To produce reproducible hematoxylin and eosin stained sections with crisp nuclear detail and clear cytoplasmic contrast, suitable for routine histopathology reporting.

2. Scope

Applies to routine H&E staining of paraffin sections, manual or automatic. Special stains are covered by their own SOPs.

3. Responsibility

• Histotechnician — performs staining, monitors reagent condition and records batches.
• Senior technician — approves protocols, authorises reagent changes and checks stain quality daily.
• Pathologist / quality manager — approves this SOP and reviews staining quality trends.

4. Equipment & materials

• Automatic slide stainer (or manual staining set) with a validated H&E protocol
• Xylene/substitute, graded alcohols, hematoxylin, differentiator (acid alcohol), bluing reagent, eosin
• Adhesive/charged slides for difficult tissue; mounting medium and coverslips
• Reagent-change log and staining register

5. Safety precautions

• Solvents and stains are hazardous — ventilated area, PPE and correct disposal.
• Keep hematoxylin filtered and covered to reduce surface oxidation/precipitate.

6. Procedure

Deparaffinise & rehydrate

1. Remove paraffin in xylene/substitute, then rehydrate through descending graded alcohols to water.
2. Ensure deparaffinisation is complete — residual wax blocks aqueous staining. Bath times: [lab to validate].

Nuclear stain

1. Stain nuclei in hematoxylin. Time: [lab to validate] (depends on hematoxylin type and freshness).
2. Rinse, then differentiate briefly in acid alcohol to sharpen nuclear detail — avoid over-differentiation.
3. Blue the sections adequately (bluing reagent or running water). Time: [lab to validate].

Counterstain & finish

1. Counterstain cytoplasm in eosin. Time: [lab to validate]; avoid over-rinsing which strips eosin.
2. Dehydrate through ascending alcohols, clear in xylene/substitute and coverslip promptly (do not let sections dry before mounting).

7. Quality-control checkpoints

• Nuclei are crisp blue-purple with visible chromatin detail — not pale (under) or inky (over).
• Cytoplasm and connective tissue show clear pink eosin contrast.
• No precipitate, no blue background, no tissue lifting.

8. Acceptance criteria

• A control/known section shows balanced nuclear and cytoplasmic staining.
• No recurring staining defects across the batch.

9. Common errors & corrective action

10. Records & documentation

• Staining register (date, batch, protocol, operator)
• Reagent-change / filtration log
• Stain-quality / corrective-action record

11. Review frequency

Review at least annually, or after any reagent, protocol or equipment change, or a recurring defect.

12. Approval