Histology Fixatives

Fixation: Purpose and Importance

• The purpose of fixation is to preserve tissues in a life-like state as possible.
• Fixation should be carried out as soon as possible after tissue removal or death to prevent autolysis.

Types of Fixatives

• Aldehydes:

  • Include formaldehyde (formalin) and glutaraldehyde.
  • Fix tissues by forming cross-linkages between proteins, particularly between lysine residues.
  • Formaldehyde:
    • Good for immunohistochemical techniques due to minimal protein structure harm.
    • Penetrates tissue well, but is relatively slow.
    • Standard solution: 10% neutral buffered formalin.
  • Glutaraldehyde:
    • Causes deformation of alpha-helix structure in proteins, making it poor for immunohistochemical staining.
    • Fixes quickly, making it good for electron microscopy.
    • Penetrates poorly, but gives best overall cytoplasmic and nuclear detail.
    • Standard solution: 2% buffered glutaraldehyde.

• Mercurials:

  • Fix tissues by an unknown mechanism.
  • Contain mercuric chloride and include B-5 and Zenker's fixatives.
  • Penetrate relatively poorly, cause some tissue hardness, but are fast and give excellent nuclear detail.
  • Best application: fixation of hematopoietic and reticuloendothelial tissues.
  • Must be disposed of carefully due to mercury content.

• Alcohols:

  • Include methyl alcohol (methanol) and ethyl alcohol (ethanol).
  • Are protein denaturants and not used routinely for tissues due to brittleness and hardness.
  • Good for cytologic smears due to quick action and good nuclear detail.

• Oxidizing Agents:

  • Include permanganate fixatives (potassium permanganate), dichromate fixatives (potassium dichromate), and osmium tetroxide.
  • Cross-link proteins, but cause extensive denaturation.
  • Have specialized applications, but are used infrequently.

• Picrates:

  • Foremost among these is Bouin's solution.
  • Has an unknown mechanism of action.
  • Does almost as well as mercurials with nuclear detail, but does not cause as much hardness.
  • Picric acid is an explosion hazard in dry form and stains skin and surfaces yellow.

Factors Affecting Fixation

• Fixation is best carried out close to neutral pH, in the range of 6-8.
• Hypoxia of tissues lowers pH, requiring buffering capacity in the fixative to prevent excessive acidity.
• Common buffers include phosphate, bicarbonate, cacodylate, and veronal.
• Penetration of tissues depends on fixative diffusability, with formalin and alcohol penetrating best, and glutaraldehyde worst.
• Sectioning tissues thinly (2-3 mm) helps with penetration.
• Volume of fixative is important, with a recommended 10:1 ratio of fixative to tissue.