Hematoxylin-Eosin Staining

Hematoxylin-Eosin Staining

Hematoxylin-Eosin (H&E) staining has been used for more than a century and is still essential for identifying various tissue types and the morphological changes that form the basis of modern cancer diagnosis. For routine diagnosis, pathologists prefer H&E when observing the details of cell and tissue structure. H&E can display a wide range of cytoplasm, nucleus and extracellular matrix characteristics, for this reason, almost all teaching texts use H&E images. Hematoxylin has a dark blue-violet color and dyes nucleic acids through complex reactions. Eosin is pink and non-specifically stains proteins. In a typical tissue, the nucleus is blue, while the cytoplasm and extracellular matrix are pink to varying degrees. Well-fixed cells show considerable intranuclear detail. The nucleus exhibits different cell type and cancer type-specific heterochromatin condensation patterns (hematoxylin staining), which is very important for diagnosis.

Examples of hematoxylin and eosin staining of healthy tissues (A), ulcerative colitis (B), adenomas (C) and adenocarcinomas (D)Figure 1. Examples of hematoxylin and eosin staining of healthy tissues (A), ulcerative colitis (B), adenomas (C) and adenocarcinomas (D) (Cammarota, et al. 2010).

The H&E staining is one of the staining methods often used in paraffin section technique, which can help scientists to see the transparent characteristics of all cells of different tissue types under a microscope, and obtain the physiological and pathological information based on it. CD BioSciences has advanced experimental equipment and professional team to provide you with high-quality H&E staining and subsequent comprehensive data analysis services. If you have any needs, please feel free to contact us.

Comparison of Different Types of H&E Staining

  Progressive staining Regressive staining
Differentiator Traditionally no differentiator is used to remove excess hematoxylin Differentiator is used to aggressively remove excess hematoxylin
Background staining Probably happens Not easy to occur
Hematoxylin staining Tissue is stained with hematoxylin only to a point Tissue is overstained with hematoxylin
Commercially available hematoxylins Harris, Mayers, Gill I, II, III, proprietary formulas
Commercially available eosins Alcoholic eosins, aqueous eosins, eosins with phloxine

General Procedure of H&E Staining

Remove the wax: The first step in performing an H&E stain is to dissolve all the wax away with xylene (a hydrocarbon solvent).

Hydrate the section: After thorough de-waxing, the slide is passed through several changes of alcohol to remove the xylene, then thoroughly rinsed in water.

Apply the hematoxylin nuclear stain: The slide is now stained with a nuclear stain, which consists of a dye (oxidized hematoxylin or hematein) and a mordant or binding agent (an aluminum salt) in the solution.

Complete the nuclear stain by "blueing": 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.

Remove excess background stain: A weak acid alcohol is used to remove non-specific background staining. After this treatment, blueing and thorough rinsing again. Staining methods that include a destaining or differentiation step are referred to as "regressive" stains.

Apply the eosin counterstain: Stain the section with an aqueous or alcoholic solution of eosin. These colors many nonnuclear elements in different shades of pink.

Rinse, dehydrate, clear and mount (apply Ccver glass): After eosin staining, the slides were changed with alcohol several times to remove all traces of water, and then rinsed in several baths of xylene to "remove" the tissue and make it completely transparent. Apply a thin layer of polystyrene mountant and then a glass coverslip. If the staining and all subsequent steps are performed correctly, the slide will show all important microscopic components and remain stable for many years.

Applications of H&E Staining

  • Checking tissue structure and cell type
  • Clinical diagnosis, pathology and cancer research
  • Detect the presence of certain microorganisms in the sample
  • Being used as a control for all immunohistochemical staining to show the tissue was correctly processed and contains no artifacts
  • When using an antibody to detect a specific protein through immunohistochemistry, H&E is used to simultaneously visualize the cells where the protein is being detected


  1. Cammarota, Rosaria, et al. "The tumor microenvironment of colorectal cancer: stromal TLR-4 expression as a potential prognostic marker." Journal of translational medicine 8.1 (2010): 1-16.
  2. Cardiff, Robert D., Claramae H. Miller, and Robert J. Munn. "Manual hematoxylin and eosin staining of mouse tissue sections." Cold Spring Harbor Protocols 2014.6 (2014): pdb-prot073411.

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