Hematoxylin & Eosin Staining

Overview of Stain

H&E stain is a general tissue stain used to initial visualization of microscopic structures.

• Primary Stain: Hematoxylin
  • Demonstrates nuclei (blue)
• Counterstain: Eosin
  • Demonstrates cytoplasmic materials
  • Red blood cells (red)
  • Muscle (dark pink)
  • Collagen (light pink)

Staining Procedures

This stain may be applied either progressively or regressively.

H&E (Progressive)

Procedure	Time	Rationale	Troubleshooting

H&E (Regressive)

Procedure	Time	Rationale	Troubleshooting

Differentiation & Blueing

Differentiation is completed in regressive methods, where staining is gradually removed and proper colour is checked microscopically. The differentiation process uses acid alcohol.

• Acid alcohol contains H+ ions that compete with the mordant for tissue. Weakly bound tissues lose stain more readily than tissues that are bound more tightly to the mordant.
• This process causes discolouration (red colour) that needs to be reversed with an alkaline reagent.
  • Blueing process reverses this change.
    • Blueing reagents include: alkaline running tap water, lithium carbonate, dilute ammonium hydroxide, and Scott's Tap Water Substitute.

Troubleshooting

Nearly all steps of H&E staining can be troubleshooted as they dyes can be removed by use of acid alcohol and reapplied.

Preventative Measures

Check reagents before use:

• Hematoxylin should appear dark burgundy-purple with no sheen
  • If metallic sheen or brown/red-brown appearance, may be over-oxidized and needs replacement
  • Oxidation occurs with exposure to oxygen or as reagent gets old
• Filter all coloured reagents to remove precipitate