Hematoxylin and Eosin (H&E) staining

Ms. Sangeeta Gourh

Hematoxylin and Eosin (H&E) staining

Hematoxylin and Eosin (H&E) staining is histology’s most widely used method. It highlights cellular and tissue structure, enabling the identification of nuclei, cytoplasm, and extracellular components under a microscope.

  1. Principle of H&E Staining

  • Hematoxylin: A basic dye that binds to acidic (basophilic) structures, staining them blue or purple. The primary target is the cell nucleus, which binds to DNA, RNA, and ribosomes.
  • Eosin: An acidic dye that binds to basic (acidophilic) structures, staining them pink or red. It stains the cytoplasm, collagen, muscle fibres, and extracellular matrix.

  1. Requirements for H&E Staining

  • Reagents:
    • Hematoxylin stain
    • Eosin stain
    • Xylene or xylene substitute (for clearing)
    • Graded alcohol solutions (for dehydration)
    • Distilled water
    • Mounting medium (e.g., DPX or Canada balsam)
    • Coverslips
  • Equipment:
    • Microscope slides
    • Coverslips
    • Staining jars or automated stainer
    • Forceps
    • Pipette or dropper
    • Drying oven (optional)

  1. Procedure for H&E Staining

Preparation and Staining

  1. Deparaffinization and Hydration:
    • Place the slide with the paraffin-embedded tissue in xylene to remove the paraffin.
    • Rehydrate through graded alcohol solutions (100%, 95%, 70%) and finally distilled water.
  2. Staining with Hematoxylin:
    • Immerse the slide in hematoxylin solution for 5–10 minutes.
    • Rinse with tap water to remove excess stains.
    • Differentiate in a weak acid solution (e.g., 0.5% hydrochloric acid in alcohol) for a few seconds to remove excess hematoxylin, enhancing nuclear contrast.
    • Blueing: Rinse the slide with an alkaline solution or tap water to shift the hematoxylin colour from red to blue.
  3. Staining with Eosin:
    • Immerse the slide in eosin for 1–3 minutes.
    • Rinse with tap water to remove excess stains.
  4. Dehydration and Clearing:
    • Dehydrate through a graded series of alcohol solutions (70%, 95%, 100%) to remove water.
    • Clear in xylene to make the tissue transparent.

Mounting and Mounting Media

  1. Application of Mounting Medium:
    • Place a drop of non-aqueous mounting medium (e.g., DPX or Canada balsam) on the tissue section.
    • Carefully place a coverslip on the slide to avoid air bubbles.
  2. Drying:
    • Allow the mounting medium to dry, securing the tissue and coverslip.
  1. Results of H&E Staining
  • Nuclei: Appear blue to purple, easily identifying nuclear structure and cellular arrangement.
  • Cytoplasm: Stains shades of pink, highlighting different types of cells and tissues.
  • Connective Tissue: Stains light pink to red, aiding in identifying extracellular matrix and fibrous structures.
  • Muscle Fibers: Usually appear pink, making them distinguishable from other tissue types.

 

  1. Dye Chemistry and Classification in Staining

Dye chemistry is based on the interaction between dyes and cellular structures. These dyes generally fall into different classes depending on their charge, solubility, and staining characteristics:

  • Acid Dyes: These dyes carry a negative charge (anionic) and stain positively charged, basic structures in cells (acidophilic or eosinophilic regions). They often colour structures like the cytoplasm and extracellular proteins.
    • Example: Eosin binds to the basic components of cells and stains them in pink to red shades.
  • Basic Dyes: Carry a positive charge (cationic) and stain negatively charged structures like nucleic acids in the nucleus and ribosomes in the cytoplasm.
    • Example: Hematoxylin (after oxidation to haematein and combined with a mordant) binds acidic components, particularly nucleic acids, staining them blue or purple.
  • Neutral Dyes: Contain both acidic and basic components, allowing them to stain both acidic and basic structures, giving a polychromatic appearance (i.e., multiple colours). These are less commonly used but are found in some complex staining procedures.
  • Indifferent Dyes are lipid-soluble and used primarily to stain fats and lipids. They rely not on ionic bonding but on solubility and physical embedding into lipids.
    • Example: Sudan Black stains lipid structures due to its solubility in lipids rather than ionic interactions.

Dye Interaction Mechanisms

The primary mechanism of staining involves either electrostatic attraction (ionic bonding) or physical embedding within cell structures:

  • Electrostatic Binding: Dyes like hematoxylin and eosin are attracted to cellular components with opposite charges, creating a strong bond that leads to specific staining patterns.
  • Hydrogen Bonding: Some dyes form hydrogen bonds with tissue structures, contributing to more specific staining properties.
  • Lipophilic Interactions: Nonpolar dyes, like those used in fat staining, embed within cell membranes or lipid droplets based on solubility rather than charge.

 

  1. Theory of Staining

The theory behind staining largely involves how dyes interact with tissues and the role of tissue chemistry:

  • Physical Theory: Suggests that dye molecules are absorbed by tissues based on permeability and size. Fat-soluble dyes stain lipid-rich areas since they penetrate and dissolve into lipid-rich structures.
  • Chemical Theory: Based on chemical bonding, where dyes form specific bonds with tissue molecules. This can include ionic, covalent, and hydrogen bonds, making the dye-tissue interaction stable and specific.
  • Electrostatic Theory: One of the most accepted theories, explaining the differential binding of basic and acidic dyes based on opposite charges. This is the basis for the widespread use of basic and acidic stains in histology.

 

  1. Role of Solvents in Staining

Solvents are crucial for preparing staining solutions, removing excess dye, and in tissue processing steps like dehydration and clearing:

  • Water: Used in water-soluble dye solutions, especially for hydrophilic tissues or in cases where dehydration is not needed.
  • Alcohol: Used in the dehydration process and for alcohol-soluble dyes. Ethanol or methanol is often used with staining to prepare slides for non-aqueous mounting media.
  • Xylene and Xylene Substitutes: Used as a clearing agent post-staining to remove alcohol and make tissues transparent. This is important for brightfield microscopy.
  • Acetone: Sometimes used as a fast-drying solvent in dehydration steps or to remove lipids from tissues before staining.

 

  1. Mordants in Staining

Mordants enhance dye binding by forming a stable, often insoluble complex with the dye, which binds strongly to the tissue. This increases staining intensity and specificity:

  • Function of Mordants: Mordants bridge the dye and the tissue, allowing dyes to bind more strongly. They are often metal ions that form dye-metal complexes, creating a more intense and stable colouration.
  • Examples of Common Mordants:
    • Aluminum Salts (e.g., aluminium sulfate): Commonly used in hematoxylin stains to create a blue colour and bind tightly to the nucleus.
    • Iron Salts (e.g., ferric chloride): Used in iron hematoxylin stains, producing intense, dark blue-black staining, often for nuclear and muscle fibres.
    • Potassium Alum: Common in traditional hematoxylin stains, it acts as a weakly acidic mordant that shifts hematoxylin to a blue stain upon “blueing.”

 

  1. Accelerators and Accentuators

Accelerators and accentuators modify the staining rate and strength by altering the solution’s pH, ionic concentration, or polarity.

  • Accelerators: These agents increase the staining rate by adjusting the solution’s pH or facilitating dye penetration. For example, alkaline solutions (such as ammonia water) can accelerate hematoxylin “blueing,” converting hematoxylin-stained areas from red to blue.
  • Accentuators: Accentuators enhance dye uptake or intensify staining by optimizing conditions for dye binding.
    • Acetic Acid: Lowers the pH of eosin solutions, allowing for a more intense, vibrant pink color in cytoplasmic staining.
    • Phenol: Occasionally used as an accentuator to increase dye penetration into hard tissues like keratin or myelin.

 

  1. Practical Application of H&E Staining with These Components

In H&E staining, all these factors come into play in a sequence:

  1. Deparaffinization: Removes wax using xylene, then rehydration through graded alcohol to distilled water.
  2. Staining with Hematoxylin:
    • Hematoxylin used with an aluminium mordant, binds to the acidic nuclear material.
    • Blueing step: An alkaline solution (e.g., ammonia water or Scott’s tap water) accelerates the blueing of hematoxylin, shifting the colour from red to blue for stable nuclear staining.
  3. Staining with Eosin:
    • Eosin, an acidic dye, binds to basic cellular components, staining cytoplasm and connective tissues in pink shades.
    • Acetic acid can be added as an accentuator to eosin to enhance staining intensity.
  4. Dehydration and Clearing:
    • Graded alcohol series (70% to 100%) dehydrates the tissue, followed by xylene to make it clear and suitable for mounting.
  5. Mounting:
    • A mounting medium, typically a resin-based medium like DPX or Canada balsam, secures the coverslip and enhances tissue preservation.

 

Leave a Reply

Your email address will not be published. Required fields are marked *