Histology Fixatives

Study Notes

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.