Introduction

The hematoxylin and eosin stain (H&E) is the most widely used histological stain. Its popularity is based on its comparative simplicity and ability to demonstrate clearly an enormous number of different tissue structures. Hematoxylin can be prepared in numerous ways and has a widespread applicability to tissues from different sites. Essentially, the hematoxylin component stains the cell nuclei blue-black, showing good intranuclear detail, while the eosin stains cell cytoplasm and most connective tissue fibers in varying shades and intensities of pink, orange, and red. While automated staining instruments and commercially prepared hematoxylin and eosin solutions are more commonly used in today’s laboratories for routine staining, students of histological techniques should have a basic knowledge of the dyes and preparation techniques in order to troubleshoot and/or modify procedures for specialized use. It should be noted that hematoxylin has additional uses beyond just the hematoxylin and eosin combination.

Eosin

Eosin is the most suitable stain to combine with an alum hematoxylin to demonstrate the general histological architecture of a tissue. Its particular value is its ability, with proper differentiation, to distinguish between the cytoplasm of different types of cell, and between the different types of connective tissue fibers and matrices, by staining them differing shades of red and pink.

The eosins are xanthene dyes and the following types are easily obtainable commercially: eosin Y (eosin yellowish, eosin water-soluble) C.I. No. 45380 (C.I. Acid Red 87); ethyl eosin (eosin S, eosin alcohol-soluble) C.I. No. 45386 (C.I. Solvent Red 45); eosin B (eosin bluish, erythrosin B) C.I. No. 45400 (C.I. Acid Red 91).

Of these, eosin Y is much the most widely used, and despite its synonym it is also satisfactorily soluble in alcohol; it is sometimes sold as ‘water and alcohol soluble’. As a cytoplasmic stain, it is usually used as a 0.5 or 1.0% solution in distilled water, with a crystal of thymol added to inhibit the growth of fungi. The addition of a little acetic acid (0.5 ml to 1000 ml stain) is said to sharpen the staining. Differentiation of the eosin staining occurs in the subsequent tap water wash, and a little further differentiation occurs during the dehydration through the alcohols. The intensity of eosin staining, and the degree of differentiation required, is largely a matter of individual taste. Suitable photomicrographs of H&E-stained tissues are easier to obtain when the eosin staining is intense and the differentiation slight (at least double the routine staining time is advisable). Ethyl eosin and eosin B are now rarely used, although occasional old methods specify their use: for example, the Harris stain for Negri bodies. Alternative red dyes have been suggested as substitutes for eosin, such as phloxine, Biebrich scarlet, etc.; however, although these substitutes often give a more intense red color to the tissues, they are rarely as amenable to subtle differentiation as eosin and are generally less valuable.

Under certain circumstances eosin staining is intense and difficulty may be experienced in obtaining adequate differentiation; this may occur after mercuric fixation. Over-differentiation of the eosin may be continued until only the red blood cells and granules of eosinophil polymorph are stained red. This is, occasionally, used to facilitate the location and identification of eosinophils. Combining eosin Y and phloxine B (10 ml 1% phloxine B, 100 ml 1% eosin Y, 780 ml 95% alcohol, 4 ml glacial acetic acid) produces a cytoplasmic stain, which more dramatically demonstrates various tissue components. Muscle is clearly differentiated from collagen, and red cells stain bright red. According to Luna (1992), eosin dye content should be 88% and not contain sodium sulfate (sometimes used as filler). When using this dye in solution, a fine granular precipitate forms, and the staining of the cytoplasm will be poor.

Hematoxylin

Hematoxylin is extracted from the heartwood (‘logwood’) of the tree Hematoxylon campechianum that originated in the Mexican State of Campeche, but which is now mainly cultivated in the West Indies. The hematoxylin is extracted from logwood with hot water, and then precipitated out from the aqueous solution using urea (see prior editions). Hematoxylin itself is not a stain. The major oxidization product is hematein, a natural dye that is responsible for the color properties. Hematein can be produced from hematoxylin in two ways.

Alum hematoxylins

This group comprises most of those that are used routinely in the hematoxylin and eosin stain, and produce good nuclear staining. The mordant is aluminum, usually in the form of ‘potash alum’ (aluminum potassium sulfate) or ‘ammonium alum’ (aluminum ammonium sulfate). All stain the nuclei a red color, which is converted to the familiar blue-black when the section is washed in a weak alkali solution. Tap water is usually alkaline enough to produce this color change, but occasionally alkaline solutions such as saturated lithium carbonate, 0.05% ammonia in distilled water, or Scott’s tap water substitute (see Appendix III) are necessary. This procedure is known as ‘blueing’.

The alum hematoxylins can be used regressively, meaning that the section is over-stained and then differentiated in acid alcohol, followed by ‘blueing’, or progressively, i.e. stained for a predetermined time to stain the nuclei adequately but leave the background tissue relatively unstained. The times for hematoxylin staining and for satisfactory differentiation will vary according to the type and age of alum hematoxylin used, the type of tissue, and the personal preference of the pathologist. For routine hematoxylin and eosin staining of tissues, the most commonly used hematoxylins are Ehrlich’s, Mayer’s, Harris’s, Gill’s, Cole’s, and Delafield’s. Carazzi’s hematoxylin is occasionally used, particularly for urgent frozen sections.

Ehrlich’s hematoxylin, being a strong hematoxylin solution, stains nuclei intensely and crisply, and stained sections fade much more slowly than those stained with other alum hematoxylins. It is particularly useful for staining sections from tissues that have been exposed to acid. It is suitable for tissues that have been subjected to acid decalcification or, more valuably, tissues that have been stored for a long period in formalin fixatives which have gradually become acidic over the storage period, or in acid fixatives such as Bouin’s fixative. Ehrlich’s hematoxylin is not ideal for frozen sections.

As with most of the chemically ripened alum hematoxylins, the quality of the nuclear staining begins to deteriorate after a few months. This deterioration is marked by the formation of a precipitate in the stored stain. At this stage, the stain should be filtered before use, and the staining time may need to be increased. For the best results, it is wise to prepare a fresh batch of stain every month, although this may be uneconomical unless only small quantities are prepared each time.

Like Mayer’s hematoxylin, Carazzi’s hematoxylin may be used as a progressive nuclear counterstain using a short staining time, followed by blueing in tap water. It is particularly suitable, since it is a pale and precise nuclear stain and does not stain any of the cytoplasmic components.

Double or triple hematoxylin concentrations may be used as preferred. These are usually referred to as Gill’s I (normal), Gill’s II (double), and Gill’s III (triple), with the Gill III being the most concentrated. Gill’s hematoxylin is more frequently used for routine H&E staining than Mayer’s hematoxylin, and is more stable than Harris’s hematoxylin, as auto-oxidation is inhibited to the extent that no measurable changes occur over many months. Disadvantages associated with Gill’s hematoxylin include staining of gelatin adhesive and even the glass itself. Some mucus may also stain darkly, as compared to Harris’s, where mucus generally remains unstained, and the glass usually fails to attract the stain. Feldman and Dapson (1987) theorized that the aluminum sulfate mordant is responsible. Certain charged sites in the tissue, in the adhesive, and on the glass are masked by the Harris mordant, leaving them unavailable for staining. Gill’s mordant system fails to do that, and the sites attract the dye-mordant complex.

Staining times with alum hematoxylins

It is not possible to give other than a rough guide to suitable staining times with alum hematoxylins because the time will vary according to the following factors:

1. Type of hematoxylin used, e.g. Ehrlich’s 20–45 minutes, Mayer’s 10–20 minutes.

2. Age of stain. As the stain ages, the staining time will need to be increased.

3. Intensity of use of stain. A heavily used hematoxylin will lose its staining powers more rapidly and longer staining times will be necessary.

4. Whether the stain is used progressively or regressively, e.g. Mayer’s hematoxylin used progressively 5–10 minutes, used regressively 10–20 minutes.

5. Pre-treatment of tissues or sections, e.g. length of time in fixative or acid decalcifying solution, or whether paraffin or frozen sections.

6. Post-treatment of sections, e.g. subsequent acid stains such as van Gieson.

7. Personal preference.

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