What is haematoxylin and exosin (H & E) staining?

November 12, 2012

What is haematoxylin and exosin (H & E) staining?

What is haematoxylin and exosin (H & E) staining?H & E staining is a complex method used in medical diagnosis.

Histology is the microscopic examination of cell tissue structure, haematoxylin and exosin stain is the most widely used stain used in medical diagnosis. Stain is used to identify the area of a biopsy or other sample which is to be examined by the pathologist – this area will generally be labeled as the HE section.

Staining method

The haematoxylin and exosin staining method involves a complex called hemalum – a complex formed from aluminum ions and oxidized hematoxylin. Once the hemalum is applied, the nuclei of cells in the sample will be colored blue. When the nuclei staining is finished a solution of eosin Y will be applied which colors the rest of the eosinophilic structures shades of red, pink and orange.


The histologist using haematoxylin and exosin has a range of protocols to choose from, generally they can be used interchangeably, and choice is dependent upon the needs of the pathologist. Differences occur in -

  • Dye composition

  • Staining protocol

  • Intensity of the blue dye

The stain contrast for individual tissue will vary according to the approach followed.


Haematoxylin solutions, when used in histological studies, are regularly used to stain the nuclei of cells blue. They may also be used in the same way for things such as keratohyalin granules. Lakes or colored complexes are formed by the use of mordants such as alum and iron – these will demonstrate nuclear and cytoplasm structures. The color of the resulting lake is dependent on the salt used – aluminum producing a blue lake; ferric producing a blue-black result. There are three main hematoxylin solutions are -

  • Ehrlich’s haematoxylin

  • Harris’s haematoxylin

  • Mayer’s haematoxylin

Eosin solutions – eosin is generally used as a counter stain in haematoxylin and exosin staining. Eosin will stain red blood cells to a deep, intense redness. The coloring is the result of bromine on fluorescein. The eosin stain is acidic and shows in the more basic parts of cell structure such as the cytoplasm. Any appearance of other colors in the sample is generally caused by intrinsic pigments such as melanin.

Heamalum or haematoxylin

Whilst these names are used interchangeably, heamalum is actually a better choice in the work of haematoxylin and exosin. Haematein is the compound which combines with aluminum ions in the formation of the active dye metal complex. This haematein is the result of haematoxylin oxidation. When alum haematoxylin are used the cell nuclei will initially appear to have a light, transparent stain which then turns almost immediately blue when exposed to a neutral or alkaline substance.

The procedure known as ‘blueing’ occurs when stained sections of alum haematoxylin will be passed to a neutral or alkaline solution in order to neutralize the acid and so form and insoluble blue complex.

Some samples do not react well to haematoxylin and exosin staining and an alternate must be used in order to achieve the required contrast.

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