HE - Hematoxylin and Eosin
The deep purple-blue of hematoxylin and the warm pink of eosin form the most widely used staining technique in the world.
The dye Hematoxylin wasn't invented in a lab, it comes from nature, extracted from the heartwood of the logwood tree (Haematoxylum campechianum), native to Central America. For centuries, this tree was prized by dye-makers for producing deep blues and blacks in fabric, long before anyone thought to put it under a microscope. Eosin, on the other hand, is a completely synthetic dye, developed in the 19th century and named after Eos, the Greek goddess of dawn.
How does it work?
In short: Hematoxylin stains nucleic-acid-rich structures (like the nucleus) blue-purple, while eosin stains protein-rich structures (like the cytoplasm) pink. The contrast between these two colors is what makes H&E so easy to read under the microscope.
Here's the chemistry behind it: Hematoxylin, when bound to a mordant (usually aluminum), becomes positively charged. This allows it to bind strongly to negatively charged structures, primarily DNA and RNA. That's why the nucleus, ribosomes, and rough endoplasmic reticulum (all rich in nucleic acids) stain deep blue-purple. We call this property basophilic, (literally "base-loving"), because these structures are attracted to the basic, positively charged dye. Not every negatively charged structure binds hematoxylin equally well, though. Mucins, for example, carry a negative charge on their carbohydrate (glycan) portions, but they don't bind hematoxylin nearly as strongly as nucleic acids do. This selectivity is part of why hematoxylin has remained the go-to nuclear stain for over a century, it's remarkably good at highlighting exactly what we want to see: the nucleus.
Eosin works on the opposite principle. It's a negatively charged dye, so it binds to positively charged, protein-rich structures, cytoplasm, collagen, muscle fibers, and red blood cells. We call this property eosinophilic, "eosin-loving."
Atlantic salmon brain tissue, showing small, round, densely packed basophilic nuclei with little visible cytoplasm
Atlantic salmon brain tissue, showing small, round, densely packed basophilic nuclei with little visible cytoplasm
Developing vertebral column in salmon fry, showing the notochord (center), surrounded by cartilage with chondrocytes (left/right), and flanked by skeletal muscle fibers (pink, striated tissue)
Naming by Color
Before molecular biology gave us the ability to identify structures by their function, histologists named things based on the only reliable clue they had: how the tissue reacted to stain. If a structure turned blue‑purple with hematoxylin, it was basophilic. If it turned pink‑red with eosin, it was eosinophilic. If it did both, it was amphophilic. These names stuck because the staining patterns were consistent, every lab, every microscope, (almost) same colours. A great example is the eosinophil, literally "the eosin‑loving cell." Paul Ehrlich didn't know what the cell did; he just saw that its granules grabbed eosin like crazy. The name came first. The biology came later.
In routine H&E, most structures still fall into three visual categories: pink, purple, or a mix. It's simple, but it works, and it's why histology's vocabulary still reflects the colours we see on the slide today.
Why H&E Doesn't Always Look the Same
H&E is one of the most standardized stains in pathology, yet anyone who works with it knows the truth: it doesn't always look the same. The reason is simple but often overlooked. Hematoxylin isn't a strong dye on its own. It only becomes the nuclear stain we rely on after two critical steps: oxidation and mordant binding.
First, hematoxylin must oxidize into hematein, the actual dye. Without this "ripening," you're essentially staining with coloured water. But even hematein can't bind tissue effectively until it forms a complex with a mordant, usually a metal salt. This dye–metal complex carries the positive charge that allows it to attach to the negatively charged phosphate groups in DNA and RNA. In other words: the mordant is what makes hematoxylin stick.
Different mordants create different versions of hematoxylin, aluminium, iron, tungsten, each producing its own colour tone, intensity, and binding behaviour. That's why Harris, Mayer, Gill, Weigert's, and PTAH don't look identical. Same dye, different chemistry.
Mordant Hematoxylin Type Common Use
Aluminum (alum) Harris, Mayer, Gill, Carazzi Standard H&E, crisp blue-purple nuclear staining
Iron (ferric salts) Weigert's, Heidenhain's Sharper, more precise staining, great for muscle striations, mitochondria, myelin
Tungsten (phosphotungstic acid) PTAH Fibrin and muscle fiber staining
References:
Bancroft's Theory and Practice of Histological Techniques, 8th Edition.