Hine's H&E for Block Staining
Hine’s method is designed to stain blocks of tissue with hematoxylin and eosin (H & E). The blocks are then sectioned, dried and immediately coverslipped. Numerous identical stained sections may be prepared for demonstration and educational purposes.
Materials
Hematoxylin
| Material | Amount | Function |
|---|---|---|
| Hematoxylin, 1% aqueous | 100 mL | Dye |
| Aluminum sulphate, 5% aqueous | 75 mL | Mordant |
| Lugol’s iodine | 25 mL | Oxidant |
| Acetic acid, glacial | 8 mL | Solvent |
| Glycerol | 50 mL | Stabiliser |
Eosin
| Material | Amount | Function |
|---|---|---|
| Ethyl eosin | 1 g | Dye |
| 90% ethanol | 100 mL | Solvent |
Worcester’s fluid
| Material | Amount | Function |
|---|---|---|
| Distilled water | 200 mL | Solvent |
| Mercuric chloride | 14 g | Fixing agent |
| Formalin, concentrated | 22.5 mL | Fixing agent |
| Acetic acid, glacial | 25 mL | Fixing agent |
Compounding Procedures
Hematoxylin
- Add the ingredients in the order listed.
- Filter before use.
Eosin
- Dissolve the eosin in the ethanol, and let stand for one month.
- For use, dilute with an equal volume of 90% ethanol and filter.
Worcester’s fluid
- Dissolve the mercuric chloride in the water.
- Immediately prior to use add the formalin and acetic acid.
Protocol
- Place fresh tissue blocks in Worcester’s fluid or formalin fixed tissue blocks in formol sublimate. Blocks should be no thicker than 1.5 cm. Fix overnight.
- Remove from fixative and place into 70% ethanol for one hour.
- Add Lugols iodine to 70% ethanol until dark brown, and complete removal of mercury pigment. Place the tissue into the ethanol and change three times over about 48 hours. The time varies depending on the tissue.
- Transfer to fresh 70% ethanol (no iodine) for two hours, then to distilled water for one hour. Trim the tissue to size for processing.
- Place the blocks into hematoxylin for seven days.
- Wash blocks in running tap water overnight.
- Begin dehydration with 70% and 80% ethanols for appropriate periods.
- Stain with the working eosin solution for five days.
- Dehydrate with absolute ethanol, two changes over five to eight hours.
- Blot off excess ethanol and clear in cedarwood oil.
- Blot off excess cedarwood oil and place into xylene, two changes for ten minutes each.
- Impregnate with paraffin wax under vacuum, three changes of one hour each.
- Block out and section.
- Remove wax with xylene and coverslip with a resinous medium.
Expected Results
- Nuclei – blue
- Background – shades of pink
Notes
- The method gives good results with all tissue except CNS.
- Staining of sections has not deteriorated after 18 years.
- Staining of processed tissues has not deteriorated after 18 years.
- Bouin’s fluid can replace Worcester’s fluid, but the yellow colouration should be removed by placing in sodium bicarbonate in 50% ethanol (concentration not specified) instead of the iodinated 70% ethanol.
- If needed, decalcification can be done with Gooding and Stewart’s fluid after removal of mercury pigment and before staining with hematoxylin.
- The hematoxylin and eosin solutions can be re-used.
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- Hine, Ian F., (1981)
Block staining of mammalian tissues with hematoxylin and eosin.
Stain technology, v 56, p 119
