Hematoxylin: What is it? Eosin Staining Methods, Types, Principle and Forms Used

So far, it is still the most widely used dye in medical diagnosis and is often the gold standard.

Hematoxylin and eosin (H&E) staining is one of the leading producers of stains in histology.

This stain has not changed for many years because it works well with various fixatives and displays a wide range of cytoplasmic, nuclear, and extracellular matrix characteristics.

Hematoxylin is a compound extracted from the heartwood of the firewood tree. Hematoxylin can be considered a basic dye. It is used to stain acid structures a purplish-blue.

DNA in the nucleus and RNA in ribosomes and rough endoplasmic reticulum are acidic, so hematoxylin binds to them and stains them purple.

Hematoxylin alone is not technically a dye and will not directly stain tissues.


Therefore, it must be combined with a ‘mordant,’ a compound that helps it bind to tissue to form a tissue-mordant-hematoxylin bond.

The mordant used is typically a metallic cation, such as aluminum.

Eosin is anionic and acts as an acid dye. It is negatively charged and stains the primary (or acidophilic) structures red or pink.

Most of the proteins in the cytoplasm are bare, so eosin binds to these proteins and stains them pink.

This includes cytoplasmic filaments in muscle cells, intracellular membranes, and extracellular fibers.

As the name suggests, the H&E stain combines two dyes, hematoxylin and eosin.

The tissue stained with hematoxylin and eosin is stained with pink-orange cytoplasm, and the nuclei are stained darkly, either blue or purple.

Eosin also stains red blood cells bright red.

Types of staining methods

  • Progressive staining: when the tissue stays in the stain long enough to reach the proper endpoint. The slides should be examined at different intervals to determine when the staining is optimal.
  • Back staining: In this method, the tissue is covered and discarded (differentiated) until the proper endpoint is reached.


The oxidation product of hematoxylin is hematin, and it is the active ingredient in the staining solution.

Hematoxylin is not classified as a dye since the molecule does not have a chromophore.

In situ oxidation of hematoxylin is accomplished by adding a solid oxidant to the stain, in this case, sodium iodate.

Hematin shows properties similar to the indicators, being blue and less soluble in aqueous alkaline conditions and red and more soluble in acidic alcoholic conditions.

Under acidic conditions, hematin binds to the lysine residues of nuclear histones using a bond through a metallic ion mordant, aluminum.

To ensure saturation of the chemical binding sites, the stain is applied longer than necessary, resulting in excess tissue with very unspecific background staining.

This undesirable coloration is selectively removed by controlled leaching into an acidic alcoholic solution (acid alcohol), and the process is called “differentiation.”

Complete cellular detail is obtained by contrast with the eosin mixture.

Used forms of eosin

There are three commonly used forms of eosin:

  1. Yellowish eosin (tetrabromofluorescein, disodium salt CI 45380).
  2. Bluish eosin (el dinitobromate derivative CI 45400).
  3. Eosin Alcohol Soluble (the ethyl derivative CI 45386), the former is preferred.

Color enhancement is achieved by fortifying the stain with phloxine, a chemical member of the same family as eosin (halogenated fluorescein).

The mechanism of its staining is not fully understood, but it is believed to be electrostatic.

The best visualizations for the histologist are obtained by applying the stains under acidic conditions, thus getting more intense specific colors.

Hematoxylin and eosin (H&E) staining is a well-established technique in histopathology. However, immunohistochemistry (IHC) interpretation is performed exclusively with hematoxylin and counterstaining.


Our objective was to investigate the potential of H&E as a counterstain (H & E-IHC) to allow visualization of a marker while confirming the diagnosis on the same slide.

The quality of the immunostaining and the rapid technical performance were the main criteria for selecting the final protocol.

We stained multiple diagnostic tissues with IHC class I tests with subcellular localization markers (anti-CK7, CK20, synaptophysin, CD20, HMB45, and Ki-67).

Also, double staining in prostate tissues with high molecular weight keratins / p63 (DAB detection) and p504s (alkaline phosphatase detection).

To validate the efficacy of counterstaining, we stained Canadian Immunohistochemical Quality Control (CQC) tissue microarrays with IHC class II tests (ER, PR, HER2, and p53 markers).

Interobserver and intraobserver agreements were assessed using κ statistics.

An excellent interpretation agreement of H & E-IHC was observed compared to the IHC standard of our laboratory (κ, 0.87 to 1.00) and with the reference values ​​of CQC (κ, 0.81 to 1, 00).

Interobserver and intraobserver agreement were excellent (κ, 0.89 to 1.00 and 0.87 to 1.00, respectively).