Haemotoxylin and Eosin Staining

Contributed by Luke Hammond, QBI, The University of Queensland, Australia

Haemotoxylin and eosin stain is used to stain cell nuclei blue and the cytoplasm pink/red to aid visualization of tissue structure and morphology. The staining method involves application of the basic dye haematoxylin, which stains basophilic structures, usually the ones containing nucleic acids, such as the ribosomes and the chromatin and alcohol-based acidic eosin Y, which colors eosinophilic structures, generally composed of intracellular or extracellular protein, bright pink to yellow.

Thicker sections require exposure to each solution for longer to allow the diffusion of reagents.


Absolute alcohol Xylene 0.3% Acid Alcohol (0.3% HCl in 70% ethanol) Distilled water. Mayer’s Haematoxylin (Fronine, # II007) 1% alcoholic eosin (Fronine # 11016Q) DePeX


  • Dewax sections in xylene (2 or 3 changes of 3minutes each)
  • Rehydrate in alcohol (100%, 95%, 70%) 3minutes each.
  • Stain in Mayer’s haematoxylin 30sec. Watch how stain develops and check under microscope (nuclei should be blue with a pale blue tissue matrix)
  • Wash and “blue” in running tap water.
  • In the case of high background differentiate in acid alcohol 8-12 times briefly.
  • Wash in tap water and re-blue and check under the microscope.
  • Stain with the 1%eosin solution for 10seconds.
  • Rinse in tap water to remove the excess stain.
  • Wash in 70% ethanol and check microscopically until the tissue matrix and cellular components are differentially stained.
  • Rapidly dehydrate the sections through graded ethanol clear in xylene and mount in DePeX.

This method is based, with permission, on an original protocol available here.