Hematoxylin and Eosin (H&E) Stain: Introduction, Principle, Test Requirements, Procedure, Result- Interpretation, and Keynotes

Introduction of Hematoxylin and Eosin Stain

Hematoxylin and eosin stain is abbreviated as H&E stain. It is one of the principal tissue stains used in histopathology to find out cancer from a suspected biopsy. The stain was first introduced by A. Wissowzky in 1876. The cell nuclei are stained blue and the cytoplasm and extracellular matrix are stained pink

Principle of H&E Stain

Hematoxylin and eosin (H&E)  are the principal stains applied for the demonstration of the nucleus and the cytoplasmic inclusions. Harri’s hematoxylin (primary stain) contains alum and alum acts as a mordant that stains the nucleus light blue which turns red in the presence of acid. The cell difference is carried out by treating the tissue with an acid solution. Eosin is the counter (secondary) stain that imparts pink color to the cytoplasm and extracellular matrix.

Requirements for H&E Stain

The equipment and reagents required for H&E staining are as follows-

1. Coplin jars
2. Dropping bottles (50 ml)
3. Coverslips
4. Dissecting needles, forceps, scalpels, etc.
5. Ordinary filter papers
6. Mounting media (DPX or Canada balsam mountant)
7. Slide washing tray
8. Harri’s hematoxylin stain
9. Eosin y stain
10. 0.5 % (v/v) Hydrochloric acid
11. Diluted ammonia water
12. Microscope

Hematoxylin and Eosin Staining Procedure

The hematoxylin and eosin staining procedure completes in the following steps-

1. Deparaffinization/ dewaxing
2. Rehydration
3. Staining (It completes in  Hematoxylin, Differentiation, Bluing, and Eosin)
4. Dehydration
5. Clearing and
6. Mounting.

• Deparaffinize the section:  Flame the slide on a burner and then place it in xylene for 3 – 4 minutes. Repeat xylene treatment with agitation.
• Rehydration:  Take the section to water. Hydrate the section by passing it through decreasing concentrations of alcohol i.e. 100% ( absolute alcohol), 90%, 80%, and 70% baths for 30 to 60 seconds in each of these alcohol solutions. Wash in tap water and rinse in distilled water. Drain the sections well before staining.
• Staining: Stain the section with hematoxylin solution for 3 to 5 minutes, and wash out in running tap water. Quickly dip the slide in and out of 0.5% (v/v) hydrochloric acid. Quickly wash the slide in tap water for 30 to 60 seconds. Dip the slides several times in dilute ammonia water. (The section should appear blue colored). Clean it in tap water and then rinse it with 95% alcohol. Agitate the slide in the eosin solution for 10 to 60 seconds and drain the staining solution.
• Dehydration: Put the slide in increasing the concentration of alcohol.  Initially place in 70% alcohol for 30 to 60 seconds. Place in 95% alcohol for 30 to 60 seconds.  Place in absolute alcohol (2 changes, 30 to 60 seconds each).
• Clearing: Put the slide twice in xylene for 30 to 60 seconds each.
• Mounting: Drain the excess xylene. mount it on suitable mounting media either DPX or Canada balsam with a coverslip.

Result and Interpretation of H&E Stain

The result and Interpretation of Hematoxylin and Eosin Stain are as follows-

S. No	Staining component	Retained Color
1.	Cell nuclei	Blue color
2.	Erythrocytes	Red color
3.	Muscle, connective tissue, cytoplasm	Varying shades of pink

Keynotes on Hematoxylin and Eosin Stain

Composition of Harri’s hematoxylin stain

Ingredients	Amount
Hematoxylin	20 gm
95% (v/v) Ethanol	10 ml
Potassium or Ammonium Alum	20 gm
Mercuric Oxide	0.5 gm
Distilled Water	200ml

Preparation of Harri’s hematoxylin stain:  Dissolve 20 g of hematoxylin in about 10 ml 95% (v/v) ethanol in a mortar with a pestle (a). Solvate  20 g of ammonium ( or potassium) alum, in 200 ml of hot distilled water (b).  Mix solutions ‘a’ and ‘b’ while hot and bring them quickly to boil with constant stirring. Add 0.5 g of mercuric oxide (HgO). Remove the flame and cool as rapidly as possible with the use of running tap water. Filter and store in an amber-colored bottle. This stain is stable at room temperature ( 25°C +- 5°C) for several months.

Composition of Eosin stain

Eosin Y Stain

Ingredients	Amount
Eosin Y	1.0  gm
Distilled water	80 ml
Ethanol 95% (v/v)	320 ml
Glacial Acetic Acid	0.4 ml

Preparation of Eosin Y stain

Dissolve 1.0 g of yellow eosin in about 80 ml of distilled water.  Add 320 ml 95% (v/v) ethanol. Put on  0.4 ml of glacial acetic acid. This solution is stable at room temperature (25°C +- 5°C) for several months.

• Diluted ammonia water preparation: Add 1.5 ml of 28% (v/v) strong ammonia solution in distilled water and make the final volume with agitation.
• A variety of Eosin stains are available. e.g. pink, red, orange, and yellow. Eosin yellow (Y) is the most commonly used stain in histopathology laboratories.
• Hematoxylin is extracted from the heartwood of the logwood tree. The botanical name of the logwood tree is Hematoxylum campechianum. After oxidation, it forms hematein. Hematin is a compound that forms strongly colored complexes containing metal ions, mainly iron and aluminum salts. Metal-hematein complexes use to stain the cell nuclei prior to examination under the microscope.
• Eosin comes to the Xanthene group and it is an anionic dye by nature. Anionic dyes are important for staining cytoplasm and extracellular matrix.
• (Check the differentiation by using a microscope.
  The nuclei should appear dark purple and the rest of the tissue should appear pale)

Further Reading

1. Bancroft’s Theory and Practice of Histological Techniques (6th edition)
2. https://en.wikipedia.org/wiki/H%26E_stain
3. Hematoxylin and eosin staining of tissue and cell sections. Fischer AH, Jacobson KA, Rose J, Zeller R.
4. H&E Staining Overview: A Guide to Best Practices Cindy Sampias, Geoffrey Rolls
5. https://www.histology.leeds.ac.uk/what-is-histology/H_and_E.php
6. The Science and Application of Hematoxylin and Eosin Staining Skip Brown
7. https://www.ihcworld.com/_protocols/special_stains/h&e_ellis.htm
8. https://www.labce.com/spg464557_mordants.aspx