Hematoxylin & Eosin stainingDR. SARA A.G. SEIF EL DIN

Hematoxylin Hematoxylin is a natural dye, which is extracted from the heartwood of the tree Haematoxylum campechianum, although histotechnologists are probably more familiar with the name as Hematoxylon campechianum. The genus name Hematoxylum is derived from two Greek words: haimatos which means blood, and xylon which means wood.

Leaves and flowers of H. campechianum

The hematoxylin which we buy is extracted from the heartwood of this bloodwood tree. There may be some differences in method, but one is to chip the heartwood of freshly logged trees, then boil the chips in water. An orange-red solution is obtained, which turns yellow, then black on cooling. The water is evaporated leaving crude hamatoxylin. Further purification is undoubtedly done

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Although it is common practice to use hematoxylin, it is not itself the dye. During the preparation of staining solutions hematoxylin is converted into hematein. This is usually accomplished with:

1- chemical oxidizing agents, Sodium iodate is the most commonly used oxidizing agent for this purpose (0.2g will oxidize 1g hematoxylin). Others are mercuric oxide (now strongly deprecated because it is poisonous), potassium permanganate and iodine.

2- Natural oxidizing atmospheric oxygen over time. Hematein may also be referred to as hematoxein, although it is not usually seen in histotechnology references. Hematein is not incorporated directly into most staining solutions because it continues to oxidize in solution and forms non-staining products.

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Mordants Alum

Iron

Iodine

Lead

Tungsten

Molybdenum

Without mordant

The dye is usually used in conjunction with a mordant, the two commonest being

aluminum (as ammonium or potassium alum), or iron (ferric chloride or iron alum). Other mordants are used much less frequently but include chrome alum and phosphotungstic acid. can demonstrate fibrin, muscle striations and some neuroglia fibres.

The tissue component most frequently demonstrated is nuclear chromatin using an aluminum mordant in the Hematoxylin and Eosin general oversight staining method. Using ferric salts as the mordant, it is also used for acid resistant nuclear staining, the demonstration of muscle striations and numerous other elements.

Hematoxylin and eosin Hematoxylin and eosin staining is one of the most common method used in histology and histopathology. Basophilic nuclei, bacteria, calcium, and so on are stained "blue" with hematoxylin. Eosinophilic cytoplasm, connective, and all other tissues are counterstained "red" with eosin. Hematoxylin and eosin staining is often used to detect inflammation or to determine the integrity of a tissue.

Deparaffinize and hydrate to water Mayer's hematoxylin for 15 minutes Wash in running tap water for 10minutes Counterstain with eosin from 15 seconds to 2 minutes depending on the age of the eosin, and the depth of the counterstain desired. For even staining results dip slides several times before allowing them to set in the eosin for the desired time

Dehydrate in 95% and absolute alcohols, two changes of 2 minutes each or until excess eosin is removed. Check under microscope

Clear in xylene, two changes of 2 minutes each Mount in DPX

Mounting Media Histological sections which need to be examined for any length of time or to be stored, must be mounted under a cover-slip.

There are two types of mounting media : 1. Aqueous media - Used for material which is unstained, stained for fat, or metachromatically stained.

2. Resinous media - For routine staining.

Aqueous Mounting Media There are used for mounting sections from distilled water when the stains would be decolorised or removed by alcohol and xylene, as would be the case with most of fat stains (Sudan methods). Gome stains, e.g. methyl violet, tend to diffuse into medium after mounting. This can be avoided by using Highman's medium. Aqueous mountains require addition of bacteriostatic agents such as phenol, crystal of thymol or sodium merthiolate to prevent the growth of fungi.

1. Apathy's medium (R.I- 1.52) A very useful medium for mounting sections for fluorescent microscopy.

2. Farrant's medium (R.I. 1.43) Recommended for fat stains.

3. Glycerine jelly (R.I. 1.47) An excellent routine mountant for fat stains.

4. Highman's medium (R.I. 1.52) Recommended with the metachroamtic dyes especially methyl

violet.

Resinous mounting media Natural or synthetic resins dissolved in benzene, toluene or xylene. These are purchase readymade. In case they become too viscous they may have to be diluted with xylene. Following are some of these media.

1. Canada balsam - Natural resin (R.I. - 1.52) It is used as 60% resin by weight in xylene. H.&E stained slides are fairly well preserved but basic aniline dyes tend to fade and prussian blue is slowly bleached. Slides take few months to dry.

2. D.P.X. (R.I. 1.52) Polystyrene resin dissolved in xylene as a 20% solution. It is most commonly used. 3. There are many other synthetic resins sold under various trade names e.g. Coverbond (R.I. 1.53), H.S.R. (Harlew synthetic Resin), Histoclad (R.I. - 1.54), Permount (r.I. 1.54), Pro-Texx (R.I. 1.495).

Criteria of acceptable mounting media 1. Refractive index should be as close as possible to that of glass i.e. 1.5.

2. It should not cause stain to diffuse or fade. 3. It should not be crack or appear granular on setting. 4. It should be dry to a nonsticky consistency and harden relatively quickly.

5. It should not shrink back from edge of cover-glass. 6. It should be free flowing and free bubbles.

To mount a slide, (A) Apply a single drop of mounting medium upon tissue section. (B) Hold coverslip at 45o allowing the drop to spread along the edge of the slip. (C) Let go of slip and allow

medium to spread slowly.

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