Haematoxylin Eosin (H&E) staining

Lung tissue stained with the H&E technique. Nuclei are darkly stained in this image.

Lung tissue stained with the H&E technique. Nuclei are darkly stained in this image.

H&E stain, HE stain or hematoxylin and eosin stain, is a popular staining method in histology. It is the most widely used stain in medical diagnosis; for example when a pathologist looks at a biopsy of a suspected cancer, the histological section is likely to be stained with H&E and termed H&E section, H+E section, or HE section.

The staining method involves application of hemalum, which is a complex formed from aluminium ions and oxidized hematoxylin. This colors nuclei of cells (and a few other objects, such as keratohyalin granules) blue. Materials colored blue by hemalum are often said to be basophilic, but this is an incorrect use of the word. The nuclear staining is folowed by counterstaining with an aqueous or alcoholic solution of eosin Y, which colors eosinophilic other structures in various shades of red, pink and orange.

Haematoxylin Solutions

Haematoxylin stains are commonly employed for histologic studies, often employed to color the nuclei of cells (and a few other objects, such as keratohyalin granules) blue. The mordants used to demonstrate nuclear and cytoplasmic structures are alum and iron, forming lakes or colored complexes (dye-mordant-tissue complexes), the color of which will depend on the salt used. Aluminium salt lakes are usually colored blue white while ferric salt lakes are colored blue-black.

The three main alum haematoxylin solutions employed are Ehrlich’s haematoxylin, Harris’s haematoxylin and Mayer’s haematoxylin. The name haemalum is preferable to “haematoxylin” for these solutions because haematein, a product of oxidation of haematoxylin, is the compound that combines with aluminium ions to form the active dye-metal complex. Alum haematoxylin solutions impart to the nuclei of cells a light transparent red stain which rapidly turns blue on exposure to any neutral or alkaline liquid.

Alum or potassium aluminium sulfate used as the mordant usually dissociates in an alkaline solution, combining with OH? of water to form insoluble aluminium hydroxide. In the presence of excess acid, aluminium hydroxide cannot be formed thus failure of aluminium haematoxylin dye-lake to form, due to lack of OH? ions. Hence, acid solutions of alum haematoxylin become red. During staining alum haematoxylin stained sections are usually passed on to a neutral or alkaline solution (e.g. hard tap water or 1% ammonium hydroxide) in order to neutralize the acid and form an insoluble blue aluminium haematin complex. This procedure is known as blueing.

When tap water is not sufficiently alkaline, or is even acid and is unsatisfactory for blueing haematoxylin, a tap water substitute consisting of 3.5 g NaHCO3 and 20 g MgSO4.7H2O in one liter of water with thymol (to inhibit formation of moulds), is used to accelerate blueing of thin paraffin sections. Addition of a trace of any alkali to tap or distilled water also provides an effective blueing solution; a few drops of strong ammonium hydroxide or of saturated aqueous lithium carbonate, added immediately before use, are sufficient for a 400 ml staining dish full of water. Use of very cold water slows down the blueing process, whereas warming accelerates it. In fact, the use of water below 10°C for blueing sections may even produce pink artifact discolorations in the tissue.

The staining of nuclei by hemalum does not require the presence of DNA and is probably due to binding of the dye-metal complex to arginine-rich basic nucleoproteins such as histones. The mechanism is different from that of nuclear staining by basic (cationic) dyes such as thionine or toluidine blue. Staining by basic dyes is prevented by chemical or enzymatic extraction of nucleic acids. Such extractions do not prevent staining of nuclei by hemalum.

Eosin Solutions

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Eosin is a fluorescent red dye resulting from the action of bromine on fluorescein. It can be used to stain cytoplasm, collagen and muscle fibers for examination under the microscope. Structures that stain readily with eosin are termed eosinophilic.Eosin is most often used as a counterstain to haematoxylin in H&E (haematoxylin and eosin) staining.  Eosin stains red blood cells intensely red. Eosin is an acidic dye and shows up in the basic parts of the cell, ie the cytoplasm. For staining, eosin Y is typically used in concentrations of 1 to 5 percent weight by volume, dissolved in water or ethanol. For prevention of mold growth in aqueous solutions, thymol is sometimes added. A small concentration (0.5 percent) of acetic acid usually gives a deeper red stain to the tissue.

Other colors, e.g. yellow and brown, can be present in the sample; they are caused by intrinsic pigments, e.g. melanin.

Some structures do not stain well. Basal laminae need to be stained by PAS stain or some silver stains in order to exhibit appropriate contrast. Reticular fibers also require silver stain. Hydrophobic structures also tend to remain clear; these are usually rich in fats, eg. adipocytes, myelin around neuron axons, and Golgi apparatus membranes.

Protocols

There are a large number of H&E protocols available for the histotechnologist. For most tissues, these approaches can be used interchangably, and selection of a particular protocol will be based upon the particular needs of the investigator. Primary differences are dye composition, staining protocol, and intensity of blue dye. Staining contrast for a particular tissue will differ depending upon the approach that is used.

Mayer’s Hematoxylin Protocol

Solutions

Mayer’s Hematoxylin

  1. Dissolve 50 g aluminum potassium sulfate (alum) in 1000 ml distilled water.
  2. When alum is completely dissolved, add 1 gm hematoxylin.
  3. When hematoxylin is completely dissolved, add 0.2 gm sodium iodate and 20 ml acetic acid.
  4. Bring solution to boil and cool, and filter

Staining Method

Staining times will vary based upon depth of stain requiredFor slide-mounted immunohistochemistry, counterstain tissue for 30 seconds. For H&E staining, counterstain tissue for 5 minutes.

In order to blue the stain, put slides through 4 changes of tap water, 5 minutes each.

Results

This recipe should create sharp blue nucleus staining with little background.

Harris’ Hematoxylin and Eosin (H&E) Staining Protocol

Solutions and Reagents

Acid Alcohol Solution (1%)
  • Hydrochloric acid, 1 ml
  • 70% ethanol, 50 ml
  • Mix well.

Ammonia Water Solution (0.2%)

  • Ammonium hydroxide (concentrated), 2 ml
  • Distilled water , 1000 ml
  • Mix well.

Lithium Carbonate Solution (Saturated):

  • Lithium carbonate 1.54 g
  • Distilled water 100 ml
  • Mix well.

Eosin-Phloxine B Solution

Prepare the stock solutions first, and then create the working solution as needed.

Eosin Stock Solution

  • Eosin Y, 1 g
  • Distilled water, 100 ml
  • Mix to dissolve.

Phloxine Stock Solution

  • Phloxine B, 1 g
  • Distilled water, 100 ml
  • Mix to dissolve.


Eosin-Phloxine B Working Solution

  • Eosin stock solution, 100 ml
  • Phloxine stock solution, 10 ml
  • Ethanol (95%), 780 ml
  • Glacial acetic acid, 4 ml
  • Mix well.

Hematoxylin Solution (Harris)

  • Potassium or ammonium (alum), 100 g
  • Distilled water, 1000 ml
  1. Heat to dissolve. Add 50 ml of 10% alcoholic hematoxylin solution and heat to boil for 1 minute.
  2. Remove from heat and slowly add 2.5 g of mercuric oxide (red).
  3. Heat to the solution and until it becomes dark purple color.
  4. Cool the solution in cold water bath and add 20 ml of glacial acetic acid (concentrated).
  5. Filter.

Staining Procedure

  1. Deparaffinize sections, 2 changes of xylene, 10 minutes each.
  2. Re-hydrate in 2 changes of absolute alcohol, 5 minutes each.
  3. 95% alcohol for 2 minutes and 70% alcohol for 2 miuntes.
  4. Wash briefly in distilled water.
  5. Stain in Harris hematoxylin solution for 8 minutes.
  6. Wash in running tap water for 5 minutes.
  7. Differentiate in 1% acid alcohol for 30 seconds.
  8. Wash running tap water for 1 minute.
  9. Bluing in 0.2% ammonia water or saturated lithium carbonate solution for 30 seconds to 1 minute.
  10. Wash in running tap water for 5 minutes.
  11. Rinse in 95% alcohol, 10 dips.
  12. Counterstain in eosin-phloxine solution for 30 seconds to 1 minute.
  13. Dehydrate through 95% alcohol, 2 changes of absolute alcohol, 5 minutes each.
  14. Clear in 2 changes of xylene, 5 minutes each.
  15. Mount with xylene based mounting medium.

Results

Nuclei should be blue, cytoplasm pink to red.

References

  1. Kiernan JA (2008) Histological and Histochemical Methods: Theory and Practice. 4th ed. Bloxham, UK: Scion.
  2. Lillie RD, Pizzolato P, Donaldson PT (1976) Nuclear stains with soluble metachrome mordant lake dyes. The effect of chemical endgroup blocking reactions and the artificial introduction of acid groups into tissues. Histochemistry 49: 23-35.
  3. Llewellyn BD (2009) Nuclear staining with alum-hematoxylin. Biotech. Histochem. 84: 159-177.
  4. Puchtler H, Meloan SN, Waldrop FS (1986) Application of current chemical concepts to metal-haematein and -brazilein stains. Histochemistry 85: 353-364.

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