Hematoxylin & Eosin Staining

 Dye chemistry and staining procedure

Introduction

The Hematoxylin and Eosin stain (H&E) is the most widely used histological stain because :

• comparative simplicity
• Ability to demonstrate clearly an enormous number of different tissue structures.
• Hematoxylin stains cell nuclei blue black  shows good intranuclear detail.
• Eosin stains cell cytoplasm and most connective tissue fibers in varying shades and intensities of pink, orange, and red.

Basics of Staining

• Stains >chemical substances used to achieve visible color contrast in the microscopic picture of a prepared tissue
• Staining >treating tissues or cells with a series of reagents so that it acquires a color; no particles of dye are seen and the stained element is transparent

Purpose of Staining

• Outlines tissues and cellular components.
  
  
• Identification of tissues.
• Establishes the presence or absence of disease processes.

Common Staining Method

 Most commonly used staining methods are –

• Hematoxylin and Eosin staining in Histopathology
• Gram’s Stain and Ziehl-Neelson staining in Microbiology.
• Romanowsky staining in Hematology.
• Papanicoloau staining in Cytology

Dyes:-

• Essentially Aromatic benzene ring compounds or derivatives that possess the twin properties of color and ability to bind to tissue.

Mordant

• A polyvalent metal ion which forms coordination complexes
• with certain dyes.
• A substance which acts as an intermediary between dye and tissue, thus increasing the affinity between them.
• Strictly applicable to salts and hydroxides of divalent and trivalent metals.
• The mordant dye complex ‘lake’ combines with tissue to form tissue-mordant-dye complex, which is insoluble.

Mordant - Dyes Application

• Mordant is applied first, followed by the dye.
• e.g Heidenhain’s iron hematoxylin
• Mordant and dye are mixed together and then applied.
• Commonly done in histotechnology
• e.g Alum hematoxylin solutions
•  Dye applied first, followed by the mordant. Hardly done in histotechnology.

Accentuators

Accentuators

• Substances which increase the staining power of dye.
• They increase the intensity & selectivity of stain.
• e.g KOH in Lofflers methylene blue
• phenol in carbol fuschin & carbol thionin.

Accelerators

• Accentuators used in metallic impregnation technique for the nervous system.
• e.g chloral hydrate

Trapping agents

• Agents which holds dye combination with tissue or bacteria .
• e.g tannic acid/iodine

Types of Staining Reaction

• Absorption or direct staining – tissue penetrated by dye solution.
• Indirect staining – using intermediate treatment with mordant
• Physical staining – simple solubility of dye in element of tissue.
• Chemical staining – formation of new substance e.g. PAS
• Adsorption phenomenon – accumulation on the surface of the compound.

Types of Staining Method

Vital:- Applied to living tissue

Accomplished by injecting staining solution into some part of the body

Mixing of stain with living cells

Primarily used for research.

Routine:- Stains tissues with minimal differentiation

except between nucleus and cytoplasm.  Demonstrates general relationship among  cells, tissues and organs.

e.g Hematoxylin and Eosin stains

Special :- Demonstrates selective features of tissues

:

Particular cell products.

Microscopic intracellular and intercellular  structure.

e.g PAS stain for mucopolysaccharide.

Regressive

• Tissue is initially overstained and then partially decolorized (differentiated) until the proper endpoint is reached.
• Sharper degree of differentiation is obtained
• The differentiation is controlled visually by microscopic examination.
• Faster and more convenient .

Progressive

• Tissue is stained for a predetermined time for adequate staining of the nuclei and leaves the background tissue relatively unstained.
• Once the dye is taken up by the tissues, it is not removed.
• The tissue is left in the dye solution until it retains the desired
• amount of coloration.
• The differentiation solely relies on the selective affinity of dyes for different tissue elements.

Differentiation

• Removal or washing out of excess stain until the color is retained only by the tissue component that is to be studied.
• Done with acid alcohol or ethyl alcohol
• Exposure to air may oxidize and improve the process.

Requirements for Staining

• All glassware should be thoroughly cleaned.
• Correct solvent should be used.
• Silver and osmic acid solutions should be kept in dark bottles.
• Solutions like dilute ammonia should be freshly prepared.
• Constituents of stain dissolved should follow the formula.
• Alcoholic solutions of the stain should be kept in dark stoppered bottles.
• All dyes should be filtered before use.

Eosin

Xanthine dyes which stains connective tissue and cytoplasm in varying intensity and shades (red to pink).

Available in the following types :

• Eosin Y ( Eosin Yellowish, Eosin water soluble) – most widely available.
• Ethyl Eosin (Eosin S, eosin alcohol soluble).
• Eosin B ( Eosin Bluish, Erythrosine B).

Ethyl eosin and eosin B are now rarely used, although occasional old methods specify their use – e.g the Harris stain for Negri bodies.

The Hematoxylin and Eosin staining Technique  For Paraffin Sections

• REMOVAL OF WAX.
• HYDRATION WITH GRADED ALCOHOLS.
• STAINING.
• DIFFERENTIATION
• BLUEING
• COUNTERSTAIN WITH EOSIN
• DEHYDRATION THROUGH GRADED ALCOHOL.
• CLEARING IN XYLENE
• MOUNTING UNDER A COVER SLIP.