L6 Staining Procedure In Histology Technique m2jw

L6: Staining Procedure in Histology Technique

HISTOPATHOLOGY I HIS 1213

Introduction Hematoxylin and Eosin stain Type or classification of dye used for identification of tissue Direct and indirect staining technique

Introduction • Tissues and their constituent cells are usually transparent and colour less when examined under the light microscope. • Therefore, differentiation between various structures is not noticeable

• Coloring (dyeing or staining) the sections of tissues makes it possible to see and study the physical features and relationships of the tissues their constituent cells. • Different tissues possess different tissue components of the cell, hence showing different affinities for most dyes or stains. • Affinities – tendency of a stain to transfer from solution onto a section

• Hence, no single staining method will demonstrate all the tissue structures present. • It is often necessary to carry out several different stains on consecutive sections from a block of tissue in order to make a diagnosis.

TISSUE – STAIN INTERACTION • Van der Waal’s (eg: intermolecular attractions) Occur between all reagents and tissue substrates . • Hydrogen bonding It is a dye-tissue attraction when a hydrogen atom is in between two electronegative atoms (eg: O, N). • Covalent Bonding

FACTORS INFLUENCING STAINING REACTIONS

• Physical factors • Chemical factors

• Physical factors Osmosis and capillarity (responsible for the penetration of stains into porous tissues) Absorption (Demonstrated by the action of certain stains on certain tissues in the presence of mineral salts)

Selective absorption (characteristic of certain substances to adsorb certain ions from a solution more readily than from others.)

• Chemical factors Based on the assumption that certain parts of biological tissues are acid in nature (eg: nuclei acid) while other parts are basic (eg: cytoplasm). The colouring matter in basic dyes is contained in the basic part of the compound, leaving the acidic part colourless. Vice – versa.

Methodology of Staining 1. Removal of paraffin wax (dewaxing) Paraffin wax is not permeable to stains, it is removed by xylene. 2. Removal of xylene Xylene is not miscible with watery solutions and low grade alcohol and therefore it is needed to be removed by absolute alcohol.

3. Gradual hydration with lower grade alcohol By immersion in 90% and 70% (avoid diffusion currents that cause damages.). 4. Hydration with water Sections are rinsed thoroughly in distilled water..

5. Removal of artifact pigment Artifact pigments may be present (due to fixation). Removed by saturated picric acid (removing formalin pigment)

Hematoxylin and Eosin stain • H&E stain is the most widely used histological stain. • Advantages: Simplicity (comparatively to other stainining method) Demonstrate clearly an enormous number of different tissue structures

HEMATOXYLIN • Hematoxylin component stains the cell nuclei blue black, with good intranuclear detail. • Hematoxylin itself is not a stain. • Instead, the oxidated form of hematoxylin (Hematein) is a natural dye that is responsible for the color properties.

• Hematein can be produced from hematoxylin in 2 ways: Natural oxidation (exposure to light and air) Chemical oxidation - Chemical oxidizing agents convert hematoxylin to hematein instantaneously, hence hematoxylin solutions are ready for use immediately after preparation

• In the absence of mordants, hematein have poor affinity for tissue and hence is inadequate as nuclear stain. • The most useful mordants of hematein are: Salts of ammonium Iron Tungsten • Most mordants are mixed and present in the hematoxylin staining solutions.

• Type of hematoxylin solutions: Alum hematoxylins (Mayer’s hematoxylin) (routinely used in H & E stain, produce good nuclear staining) Iron hematoxylins Tungsten hematoxylins Molybdenum hematoxylins Lead hematoxylins Hematoxylin without mordant.

EOSIN • Eosins are xanthene dyes. • Eosin component stains cell cytoplasm and most connective tissue fibers in varying shades and intensities of pink, orange and red. • It is most suitable stain to combine with an alum hematoxylin to demonstrate the general histological architecture of a tissue.

• Eosin is able to differentiate and distinguish between: Cytoplasm of different types of cell Connective tissue fibres between different types of cell • Acetic acid may be added to sharpen the staining.

• The commercially available types of Eosin: Eosin Y (most widely used) Ethyl eosin Eosin B

• The need for consistency of staining is vital to avoid difficult histological interpretation. • Hence, it is important to ensure the quality of staining. • In general, automated staining machines is able to produce accurate and consistent staining. • However, problems might arise with the hematoxylin staining.

• Factors affecting the quality of staining: Fixation Variation in processing schedules Section thickness Excessive hot plate temperature

Type of dye used for identification of tissue CONNECTIVE TISSUE STAINS • Trichrome stain general term for a number techniques for the selective demonstration of muscle, collagen fibers, fibrin and erythrocytes.

• Example: The demonstration of fibrin (Techniques : Gram – Weigert; phosphotungistic acid-hematoxylin; trichrome methods) The demonstration of muscle striations ( H & E; trichrome methods.)

Periodic acid-Schiff (PAS) technique • Is the most versatile and widely used technique for the demonstration of carbohydrates and glycoconjugates. • With PAS, we can assess the thickness of basement membrane (increase in basement membrane can be a pathological conditions eg: kidney.)

• The PAS technique is based upon the reactivity between free aldehyde groups within carbohydrates and the Schiff reagents. • In PAS technique, it involves the oxidation of 1,2 glycols within carbohydrates to form adjacent aldehydes.

• Oxidizing agents commonly used is periodic acid (HIO4) (0.5-1.0%, 5-10 mins). • Other oxidizing agents are: potassium permanganate, chromic acid, etc. • The reaction between the aldehyde and the Schiff reagent results in the formation of a bright red magenta end product.

• The intensity of the color that develops following the reaction with Schiff reagent is dependent upon the concentrations of glycol structures in the tissue.

FAT STAINS • The most common stains used to demonstrate fats/lipids are oil soluble dyes. • This group of dyes includes Sudan III, Sudan IV, oil red O and Sudan black B • Sudan black B is the most sensitive of them.

• In order to penetrate fats, the Sudans must dissolved in organic solvents (solvent cehicle). • The solvents should be sufficiently diluted to avoid extracting the lipids itself. • Example of solvents used: Ethanol Isopropanol Triethyl phosphate Propylene

• For general use, 70% of ethanol is an adequate solvent for Oil red O and Sudan black. • Besides lipids, Sudan black B stains phospholipids and neutral fats. • The stained lipids will appear in orange-red.

FEULGEN REACTION • It is the standard technique for demonstrating deoxyribose. • It involves mild acid hydrolysis (employing 1 M HCL at 60oc) which is the critical part of the method. • It causes the purine-deoxyribose bond to break, resulting the exposure of aldehydes which can be demonstrated by the use of Schiff’s reagent.

• Elements containing DNA are stained at redpurple color, whereas RNA is not demonstrated.

METHYL GREEN-PYRONIN • Demonstrating both DNA and RNA. • Staining DNA in green and cytoplasmic RNA in red. • Methyl green is impure dye containing methyl violet which can be removed by washing with chloroform. • Pure methyl green appears to be specific for DNA.

• Pyronin on the hand, binds with RNA. • The pH and concentration of the staining solution is critical. • Dehydration after staining is important as well as it will give a greener nuclear staining effect.

Mounting stained Specimens • In order to obtain best result with stained sections, the slide is mounted in a transparent medium with refractive index close to that of the glass slides. • Mounting medium is desired to protect the stained section from physical damage. • It can also avoid fading of the stain due to heat or oxidation.

• Example of mounting media: Canada Balsam Gum Damar Synthetic Resins (DPX or Kirkpatrick and Lendrum, Clarite)