Hematoxylin and eosin (H&E) stain

Principle

• Hematoxylin is oxidized to hematein and combined with a mordant (usually aluminum salts) to form a positively charged dye–metal complex that binds nuclear chromatin (phosphate groups of DNA).
• Eosin Y is an acidic (anionic) dye that stains basic proteins in cytoplasm and connective tissue.
• Progressive hematoxylins (e.g. Mayer's) stain to the desired intensity; regressive ones (e.g. Harris) overstain and are then differentiated.

Specimen

• Paraffin sections (about 3–5 µm) or frozen sections.

Reagents and equipment

• Xylene or substitute, graded alcohols, alum hematoxylin, acid-alcohol (about 0.5–1% HCl in 70% alcohol), bluing agent (Scott's tap water substitute, weak ammonia or lithium carbonate), eosin Y (with or without phloxine), resinous mountant.
• Manual staining dishes or an automated stainer and coverslipper.

Procedure

• Dewax in xylene and hydrate through graded alcohols to water.
• Stain in hematoxylin; rinse in water.
• Differentiate briefly in acid-alcohol (regressive method) to remove excess dye.
• Blue in a weak alkaline solution, which turns nuclei from red-purple to blue; rinse.
• Counterstain in eosin.
• Dehydrate through alcohols, clear in xylene and mount with a coverslip.

Results

• Nuclei: blue to purple; calcium and some bacterial colonies: blue-purple.
• Cytoplasm: shades of pink; muscle: deep pink; collagen: pale pink; RBCs: bright red-orange.

Quality control and pitfalls

• Review a control slide daily for nuclear detail and eosin balance.
• Pale nuclei: exhausted hematoxylin, too short staining or over-differentiation.
• Red-brown nuclei: inadequate bluing.
• Blue-black precipitate on sections: unfiltered hematoxylin with surface oxidation film; filter daily.
• Uneven or patchy staining: incomplete dewaxing.
• Milky or hazy slides: water carried into xylene due to poor dehydration.
• Monitor and replace reagents according to slide counts.

Clinical use

• Primary routine stain in histopathology; the basis for most tissue diagnoses.