Histology Dehydration and Clearing Techniques

Dehydration

• Purpose: to remove water from tissue samples to prevent tissue degradation and facilitate infiltration with a embedding medium
• Methods:
  • Ethanol series: 70%, 80%, 90%, 95%, and 100% ethanol
  • Acetone: used for fatty tissues or when ethanol is not effective
  • Automated tissue processors: use a combination of ethanol and acetone
• Importance:
  • Prevents tissue shrinkage and distortion
  • Allows for uniform infiltration of the embedding medium

Clearing

• Purpose: to replace dehydrating agent with a solvent that allows for infiltration of the embedding medium
• Methods:
  • Xylene: most commonly used clearing agent
  • Toluene: alternative to xylene
  • Chloroform: used in some automated tissue processors
• Importance:
  • Enables the embedding medium to penetrate the tissue evenly
  • Helps to remove any remaining dehydrating agent

Embedding

• Purpose: to support the tissue sample during sectioning and provide a rigid structure for microtomy
• Methods:
  • Paraffin wax: most commonly used embedding medium
  • Plastic resin: used for electron microscopy and special applications
  • Frozen sectioning: uses a frozen tissue sample and no embedding medium
• Importance:
  • Maintains tissue structure and morphology
  • Allows for thin sectioning and high-quality microscopy

Tissue Preparation

Dehydration

• Removes water from tissue samples to prevent degradation and facilitate embedding medium infiltration
• Methods include:
  • Ethanol series (70%, 80%, 90%, 95%, and 100%)
  • Acetone (for fatty tissues or when ethanol is ineffective)
  • Automated tissue processors (combining ethanol and acetone)
• Prevents tissue shrinkage and distortion, ensuring uniform embedding medium infiltration

Clearing

• Replaces dehydrating agents with a solvent to allow embedding medium infiltration
• Methods include:
  • Xylene (most commonly used clearing agent)
  • Toluene (alternative to xylene)
  • Chloroform (used in some automated tissue processors)
• Enables even embedding medium penetration and removes remaining dehydrating agents

Embedding

• Supports tissue samples during sectioning and provides a rigid structure for microtomy
• Methods include:
  • Paraffin wax (most commonly used embedding medium)
  • Plastic resin (for electron microscopy and special applications)
  • Frozen sectioning (uses frozen tissue samples with no embedding medium)
• Maintains tissue structure and morphology, allowing for thin sectioning and high-quality microscopy

Dehydration

• Dehydration removes water from tissue samples to prepare them for infiltration with a hydrophobic embedding medium.
• Methods of dehydration include:
  • Ethanol series: Gradual replacement of water with increasing concentrations of ethanol (e.g., 70%, 95%, 100%).
  • Acetone series: Similar to ethanol series, but using acetone as the dehydrating agent.
  • Automated tissue processors: Combine heat, vacuum, and solvents to dehydrate tissues.
• Importance of dehydration:
  • Prevents ice crystal formation during freezing.
  • Allows for infiltration with embedding medium.

Clearing

• Clearing removes dehydrating agents and replaces them with a solvent that allows for infiltration with the embedding medium.
• Methods of clearing include:
  • Xylene or toluene: Used to remove ethanol or acetone from dehydrated tissues.
  • Chloroform: Used to clear tissues, especially for paraffin wax embedding.
  • Benzene: Used to clear tissues, but has largely been replaced by safer alternatives.
• Importance of clearing:
  • Solvents used in clearing must be compatible with the embedding medium.
  • Ensures complete removal of dehydrating agents.

Embedding

• Embedding surrounds the tissue sample with a medium that provides support and allows for sectioning.
• Methods of embedding include:
  • Paraffin wax: Most commonly used embedding medium, provides a firm matrix for sectioning.
  • Plastic resin: Used for electron microscopy, provides a more stable and durable matrix.
  • Gelatin: Used for embedding small tissue samples, especially for frozen sectioning.
• Importance of embedding:
  • Provides a stable matrix for sectioning.
  • Allows for precise control over tissue orientation and section thickness.