An InDepth Look at the Hematoxylin & Eosin (H&E) Stain: Part 1

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The beautiful color that the H&E stain produces is not random. We all know in histology, when staining, ionic bonds are the most important type of bonds. Ions of the opposite charge are attracted to each other through electrostatic interaction. One charge is from the tissue and the other from the dye. Without going into too many details, hematoxylin is a dye extracted from the tree Haematoxylum Campechianum. Then, it is oxidized to hematein, which is the actual dye used in H&E stain. Hematein combines with a metal cation mordant (compound that helps it to link to the tissue) such as aluminum salts. Therefore, hematoxylin acts as a basic dye. It reacts with any basophilic component of the tissue, such as the nuclei, staining it blue as a result. Unlike hematoxylin, Eosin is an acidic dye that is negatively charged. It will react with the acidophilic cell component of the tissue, such as the amino group in the protein in the cytoplasm, staining it pink as a result.

Now let’s investigate what makes one hematoxylin different from others, and how they play a major part of the decision making. Gills and Mayer’s is made up of aluminum sulphate 52g, 75% water, and 25% ethylene glycol, which keeps Gills more stable, no precipitation, no filtering. Whereas Harris is made up of aluminum ammonium sulphate 90-100g, 95% water and 5% ethylene alcohol. Harris hematoxylin produces well delineated crisp nuclear staining, however due the components in Harris, it may require weekly or daily filtering to remove precipitation caused by oxidation.

There are other factors that play a role in choosing the best technique. The question of choice is very subjective. Ultimately, it depends on the pathologist’s expectations, preferences, your lab’s workflow, and tissue types. Check out part 2 of this blog next week to learn more about regressive and progressive staining, and what we do in our lab!

Fixation on Histology