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.
| Procedure | Time | Rationale | Troubleshooting |
|---|---|---|---|
| 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.
- Blueing process reverses this change.
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