Histology and Microscopy Techniques

Fixation Methods

• Purpose: To preserve tissue morphology and prevent degradation
• Types:
  • Chemical fixation: Uses chemicals to denature proteins and preserve tissue structure
    • Formalin: most common fixative, peroxygenic acid-based
    • Methanol: used for delicate tissues, can be used for DNA and RNA preservation
    • Glutaraldehyde: used for electron microscopy, preserves ultrastructure
  • Physical fixation: Uses physical methods to preserve tissue structure
    • Freeze-drying: preserves tissue structure and antigens
    • Cryofixation: rapid freezing to preserve ultrastructure

Staining Techniques

• Purpose: To enhance contrast and visibility of tissue structures
• Types:
  • Dyes: Chemical compounds that bind to specific tissue components
    • Hematoxylin and eosin (H&E): most common staining technique, stains nuclei and cytoplasm
    • Special stains: used for specific tissue components, e.g. PAS for glycogen, Masson's trichrome for collagen
  • Immunohistochemistry: Uses antibodies to detect specific proteins
    • Direct method: uses labeled antibody
    • Indirect method: uses unlabeled antibody and labeled secondary antibody
  • Histological stains: Used for specific tissue structures or components
    • Giemsa stain: used for blood cells and parasites
    • Gram stain: used for bacterial identification

Sectioning Methods

• Purpose: To obtain thin sections of tissue for microscopic examination
• Types:
  • Microtomy: Uses a microtome to cut sections
    • Rotary microtome: most common type, uses a rotating blade
    • Cryomicrotome: used for frozen sections, cuts at -20°C
  • Vibratome: Uses a vibrating blade to cut sections
    • Used for fresh tissue, preserves tissue structure
  • Laser microtomy: Uses a laser to cut sections
    • Used for delicate tissues, preserves tissue structure

Mounting Media

• Purpose: To preserve and stabilize the tissue section on the slide
• Types:
  • Aqueous mounting media: Water-based, e.g. water, glycerol, or gelatin
    • Used for temporary mounts, can be easily removed
  • Organic mounting media: Solvent-based, e.g. xylene, toluene, or DPX
    • Used for permanent mounts, provides better clarity and durability
  • Resin-based mounting media: Used for electron microscopy, e.g. Epon or Spurr's resin

Quality Control

• Importance: Ensures accurate and reliable results
• Steps:
  • Tissue selection: Selecting appropriate tissue samples for analysis
  • Fixation and processing: Verifying fixation and processing protocols
  • Staining and mounting: Verifying staining and mounting techniques
  • Microscopic examination: Verifying slide quality and accuracy of interpretation
  • Standardization: Standardizing protocols and procedures to ensure consistency and reproducibility