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H&E Staining Procedure for Hematoxylin and Eosin
- 2. INTRODUCTION • The histopathological sections that are prepared are colorless and different components cannot be appreciated. • Staining them with different color dyes having affinity od special components of tissues helps in easy identification of morphology. • H & E staining is the most frequently used stain in histology.
- 3. HEMATOXYLIN • It is extracted from bark of tree hematoylom campechianum. • Hematoxylin itself is not a stain. • It is a major oxidation product of hematin which is responsible for natural color of dye.
- 4. • Hematin can be produced from hematoxylin from two ways: • NATURAL OXIDATION • By exposure to sunlight and air,but it is along process takes 3-4 months • Solution retain the stain for longer time.
- 5. CHEMICAL OXIDATION • Occur instantaneously with short half life • Oxidation by sodium iodate most common ly used .eg –mayers hematoxylin • Oxidation by mercuric oxide eg harris hematoxylin.
- 6. • Hematin is a weak base dye having poor affinity dye for tissues and inadequate as nuclear stain with mordant. • MORDANT-Mordant is a substance which act as intermediate between dye and tissue .it is a metal with valency of 2+. • Examples are ammonium and potassium alum ferric salt.
- 7. PROPRTIES OF HEMATOXYLIN • Hematoxylin has no staining property. • Hematin with mordant such as ammonium or potassium alum forms lake which function as cationic dye and stains anionic tissue components. • Hematin in aqueous solution can be acidic or alkaline dye depending on PH.
- 8. Classification of hematoxylin solutions • Alum hematoxylin-Harris and Mayers • Iron hematoxylin –Weigerts and Verhoeffs • Tungston hematoxylin • Molybdenum hematoxylin • Lead hematoxylin
- 9. • PROGRESSIVE STAINING • When tissue is left in the stain just long enough to reach the property end point. the slide have to be examined under microscope at different intervals. • REGRESSIVE STAINING • The tissue is overstained and then restained until the end point is reached.
- 10. • Harris hematoxylin is regressive stain the over stainig is removed by acid alcohol .The removal of excess dye is called differentiation. • The hematoxylin alum gives a reddish hue to the tissues because of acidic ph. • To convert the color to final blue alkaline ph is required. • This process is called bluing.it is done either with tap water or ammonium hydroxide.
- 11. PREPARATION OF HARRIS HEMATOXYLIN INGREDIENTS • Hematoxylin-5gm • Absolut alcohol-50ml • Ammonium alum-100gm • Distilled water-1000ml • Mercuric oxide-2.5gm • Glacial acetic acid-40ml
- 12. METHOD • Dissolve the hematoxylin in absolute alcohol and ammonium alum in hot water. • Mix the 2 solutions and heat to boiling. • Remove from the flame and add mercuric oxide and cool rapidly. • Glacial acetic acid if added gives brisk nuclear staining but life of the solution is reduced. • Hence if acetic acid is to be added it should be added in working solutions.
- 13. EOSIN • Eosin is the counter stain that stains the cytoplasm rose coloured. • The most widely used eosin is eosin y( y-yellow). • It is available in either water or alcohol soluble form. • Most of the labs use the water soluble form of eosin y in an alcohol water solution.
- 14. INGRIDIENTS • Eosin y-1 gm • Distilled water-80ml • 95% alcohol-320ml • Glacial acidic acid-0.4ml
- 15. PREPARATION • Dissolve the eosin in water and then add to this 95% alcohol. • To the final mixture add few drops of acetic acid which increase the staining intensity of eosin. • When ready to use the stain should be cloudy if clear add a few drops of acetic acid. • The solution should be standardized by stainig the control slide.
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- 17. PROCEDURE /STEPS OF H and E STAINING 7/31/19
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