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SOP templateH&E Staining SOP (Template)
A ready-to-adapt template you can tailor to your laboratory. Complete the highlighted fields and validate before use.
Purpose
To produce reproducible hematoxylin and eosin stained sections with crisp nuclear detail and clear cytoplasmic contrast, suitable for routine histopathology reporting.
Scope
Applies to routine H&E staining of paraffin sections, manual or automatic. Special stains are covered by their own SOPs.
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.
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
Safety precautions
- Solvents and stains are hazardous — ventilated area, PPE and correct disposal.
- Keep hematoxylin filtered and covered to reduce surface oxidation/precipitate.
Procedure
Deparaffinise & rehydrate
- Remove paraffin in xylene/substitute, then rehydrate through descending graded alcohols to water.
- Ensure deparaffinisation is complete — residual wax blocks aqueous staining. Bath times: [lab to validate].
Nuclear stain
- Stain nuclei in hematoxylin. Time: [lab to validate] (depends on hematoxylin type and freshness).
- Rinse, then differentiate briefly in acid alcohol to sharpen nuclear detail — avoid over-differentiation.
- Blue the sections adequately (bluing reagent or running water). Time: [lab to validate].
Counterstain & finish
- Counterstain cytoplasm in eosin. Time: [lab to validate]; avoid over-rinsing which strips eosin.
- Dehydrate through ascending alcohols, clear in xylene/substitute and coverslip promptly (do not let sections dry before mounting).
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.
Acceptance criteria
- A control/known section shows balanced nuclear and cytoplasmic staining.
- No recurring staining defects across the batch.
Common errors & corrective action
| Problem | Corrective action |
|---|---|
| Weak / pale nuclei | Replace exhausted hematoxylin; reduce differentiation; ensure adequate bluing and complete deparaffinisation; cut thinner sections. |
| Over-dark nuclei | Reduce hematoxylin time and/or increase differentiation slightly; standardise on programmed times. |
| Uneven / patchy staining | Standardise agitation and reagent levels; an automatic stainer removes manual variation. |
| Precipitate on slide | Filter/skim oxidised hematoxylin and clean staining containers. |
| Tissue lifting during staining | Use adhesive/charged slides and ensure adequate slide drying; moderate agitation. |
Records & documentation
- Staining register (date, batch, protocol, operator)
- Reagent-change / filtration log
- Stain-quality / corrective-action record
Review frequency
Review at least annually, or after any reagent, protocol or equipment change, or a recurring defect.
Related equipment & guides
Author: Unimeditrek Technical Content Team · Reviewed by: Unimeditrek Biomedical Engineering & Histopathology Applications team · Last reviewed: July 2026.
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