Hematoxylin and eosin stain

Last revised by Daniel J Bell on 12 May 2020

The combined hematoxylin and eosin (H&E) stain is the most widely used stain in histology and histopathology. Hematoxylin has an intense purple-blue hue and binds to nucleic acids. Eosin has a pink hue and non-specifically stains proteins. These two stains in combination are vital for distinguishing many tissues and for making a tentative diagnosis of malignancy 1,2.

Typically, cellular nuclei stain blue (hematoxylin binds to nucleic acids), conversely the cytosol and extracellular substance demonstrate variable pink staining (eosin binds to proteins) 1. For tissue-typing, as well as the diagnosis of cancer, the hematoxylin staining of the condensated chromatin in the cell nucleus provides important information. Moreover when there is floridity of ribosomes in the cytosol there is a clear bluish hue (due to abundant RNA). Golgi bodies may be discerned by their relative lack of staining in close proximity to the cell nucleus.

Hematoxylin is sometimes used alone as a counterstain for immunohistochemical or hybridization methods.

The following is a simplification of the full methodology for a non-specialist 1:

  1. it is essential that the specimen - on a slide - used has first been fixed in an ethanol or aldehyde-based fixative
  2. initially slide is rinsed with water for 30 seconds: a vital precursor step
  3. slide is placed in a Coplin jar (a vessel designed to hold slides vertically), filled with Mayer’s hematoxylin and shaken for 30 seconds
  4. slide is washed with water for one minute
  5. slide is rinsed and shaken with 1% eosin Y reagent for 10-30 seconds
  6. specimen on the slide is then dehydrated with a double change of 95% ethanol and then a double change of 100% ethanol
  7. the ethanol itself is removed by two changes of xylene (xylene is contraindicated if plastic slides/culture dishes as it degrades plastics)
  8. 1-2 drops of mounting medium are added to the specimen on the slide, which is then overlaid a coverslip

Hematoxylin and eosin stains were first employed over 100 years ago and the basic method has not changed for many decades.

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