The H&E stain provides a comprehensive picture of the microanatomy of organs and tissues. Hematoxylin precisely stains nuclear components, including heterochromatin and nucleoli, while eosin stains cytoplasmic components including collagen and elastic fibers, muscle fibers and red blood cells. In a high-quality H&E stain, there are subtle differences in the shades of color produced by the stains, particularly eosin, and this aids in the detection and interpretation of morphological changes associated with disease.

It is important that people performing and assessing H&E stains for quality are aware of the subtleties of the stain, know what can be achieved when the stain is properly performed with high-quality reagents, and know what to look for microscopically. The maintenance of consistent, high-quality H&E stains is a fundamental requirement in histopathology laboratories.

In the following sections, the basic steps in performing an H&E stain are outlined.

Following the preparation of a paraffin section, all the elements are infiltrated with and surrounded by paraffin wax which is hydrophobic and impervious to aqueous reagents. The majority of cell and tissue components have no natural color and are not visible. The first step in performing an H&E stain is to dissolve all the wax away with xylene (a hydrocarbon solvent).

After thorough de-waxing, the slide is passed through several changes of alcohol to remove the xylene, then thoroughly rinsed in water. The section is now hydrated so that aqueous reagents will readily penetrate the cells and tissue elements.

The slide is now stained with a nuclear stain such as Harris hematoxylin, which consists of a dye (oxidized hematoxylin or hematein) and a mordant or binding agent (an aluminum salt) in the solution. Initially this stains the nuclei and some other elements a reddish-purple color.

After rinsing in tap water, the section is “blued” by treatment with a weakly alkaline solution. This step converts the hematoxylin to a dark blue color. The section can now be rinsed and checked to see if the nuclei are properly stained, showing adequate contrast and to assess the level of background stain.

On most occasions when Harris hematoxylin is employed, a differentiation (destaining) step is required to remove non-specific background staining and to improve contrast. A weak acid alcohol is used. After this treatment, blueing and thorough rinsing is again required. Staining methods that include a destaining or differentiation step are referred to as “regressive” stains.

The section is now stained with an aqueous or alcoholic solution of eosin (depending on personal preference). This colors many nonnuclear elements in different shades of pink.

Following the eosin stain, the slide is passed through several changes of alcohol to remove all traces of water, then rinsed in several baths of xylene which “clears” the tissue and renders it completely transparent. A thin layer of polystyrene mountant is applied, followed by a glass coverslip. If the stain and all the subsequent steps have been properly performed, the slide will reveal all the important microscopic components and be stable for many years.