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A Beginner’s Guide to Haematoxylin and Eosin Staining

Once a tissue specimen has been processed by a histology lab, and transferred onto a glass slide, it needs to be appropriately stained for microscopic evaluation. This is because unstained tissue lacks contrast: all of the fixed materials have a similar refractive index and a similar color. If you viewed an unstained tissue section under the microscope, everything would appear a uniform dull grey color.

 

The staining process therefore makes use of various dyes that stain particular cell components within tissues, so that you can distinguish different cell parts from each other.

Haematoxylin and Eosin

For routine examination, haematoxylin and eosin (H&E) is the stain of choice. This stain produces colors different tissue structures, which would otherwise be transparent, so that you can get a detailed view of the tissue.

As its name suggests, H&E stain makes use of a combination of two dyes – haematoxylin and eosin. The two stains were independently introduced in 1865 and 1875, respectively, by Böhmer and Fischer. In 1876, Wissowzky described their use in combination as a tissue staining method for staining different materials different colors.

Despite its simplicity, this stain has stood the test of time. Even now, over a century later, H&E remains the most frequently used tissue stain worldwide.

The Science Behind H&E

Staining does not produce color randomly; instead, the dyes exploit differences in the chemistry of the tissue to differentially color various components.

Ionic bonding is the most important type of bonding that occurs in histologic staining techniques. It involves electrostatic attraction between ions of opposite charge, one of which is fixed in the tissue, and the second of which is in the dye.

Haematoxylin alone is not technically a dye, and will not directly stain tissues. It therefore needs to be used in combination with a “mordant” – a compound that helps it link to the tissue. The mordant used is typically a metal cation, such as aluminium. Haematoxylin in complex with aluminium salts is cationic and acts as a basic dye. It is positively charged and can react with negatively charged, basophilic cell components, such as nucleic acids in the nucleus. These stain blue as a result.

Eosin is anionic and acts as an acidic dye. It is negatively charged and can react with positively charged, acidophilic components in the tissue, such as amino groups in proteins in the cytoplasm. These stain pink as a result.

H&E is a useful all-purpose stain that is also quick and easy to use, which may explain why it has stood the test of time.  What are your tips for tissue section staining?

 

Originally published on July 10, 2012. Updated and revised on August 8, 2015.

 

2 Comments

  1. joyce on September 14, 2016 at 1:52 pm

    Any suggestions on H & E staining protocol for frozen sections using automatic stainer.
    Having difficulty with uniform staining. Bleeding eosin and murkey slides.

  2. Aziz on April 27, 2016 at 12:46 pm

    Majority of labs in US buy ready to use Hematoxylin and Eosin from suppliers, however there are some labs like our where we make our own solutions. Question is raised by State Inspectors about shelf life of these solutions prepared in labs. How do I determine it? What procedures are adopted to find shelf life besides testing on control tissues? Is it possible to determine it based on some documented experiments conducted by previous workers? If so, where do I find it.
    Thanks for your time.

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