Hematoxylin Staining Protocol


Hematoxylin staining, together with eosin staining, is a fairly standard and routinely carried out procedure in any biology, medical or pathology laboratory. The term "hematoxylin staining protocol" is incorrect but is occasionally used out of habit. Hematoxylin is a weak stain when used on its own and so it is mixed with a metallic mordant (e.g., alum). Hematoxylin-eosin (H & E) staining is used to visualize cells because it stains the nucleus very efficiently. It is also capable of staining other cellular structures such as myelin, mitotic figures and muscle striations. Hematoxylin-eosin staining protocols involve a series of rinses and incubations in hematoxylin, acid-alcohols, blueing agents and then eosin.


  • This compound is actually a natural dye derived from the logwood tree. When oxidized, it forms hematein, which is the active agent that causes the staining. In hematoxylin staining protocols, the tissue sample can be stained in any number of combinations of minutes or seconds, all depending on the nature and thickness of the tissue, and whether progressive or regressive. As a guide, renal biopsies may be stained for 10 minutes while other routine tissues only require four minutes. After staining with hematoxylin, differentiation (distinguishing the staining end-point) is done by washing in acid alcohol.

Types of Hematoxylin-based Staining methods

  • Hematoxylin staining is either progressive or regressive. In progressive staining protocols, the tissue of interest is incubated in the stain for a very brief duration until the stain is easily observed under a microscope. This prevents excessive staining of the tissue. If over-staining has occurred, the tissue can be de-stained by washing in a dilute acid-alcohol mixture until the appropriate reduction in staining is obtained. This is the principle behind regressive stains.


  • This is not an essential step; however, it improves the clarity of the hematoxylin-stained nuclei, which go from a less distinct purple to a sharper blue, which contrasts very well with a counter-stain. Scott's tap water substitute is a widely available laboratory reagent that is used for rapid blueing.

Problems with Inconsistency of Hematoxylin

  • Because of its oxidative nature, the effectiveness of a hematoxylin stain depends on how many times it has been reused, and how many days it has been kept, all of which cause it to become progressively more oxidized. It is also unavoidable that some volume of water will be transferred into a hematoxylin staining preparation, therefore diluting it and rendering it less effective. It is always best to start with a freshly prepared batch, or one that has been kept in air-proof storage (e.g., by overlaying with oil)

Eosin Staining

  • In nearly all laboratories, staining with hematoxylin is done in conjunction with the use of a red counterstain known as eosin Y. Although many other red counterstains are available, none show that the staining reaction has ended quite as clearly as eosin Y does. It is dissolved in ethanol and then added to the hematoxylin-stained slide, resulting in tissues besides the nucleus being stained red.

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