What is Fixation?
Fixation is a crucial step in the preparation of biological tissues for
microscopic examination. This process preserves the tissue by halting biological processes and stabilizing cellular structures, which prevents
degradation and maintains the tissue's morphology.
Why is Fixation Important?
The primary objective of fixation is to preserve tissues in a state as close to their natural condition as possible. It prevents
autolysis (self-digestion by enzymes) and
putrefaction (decomposition by bacteria), ensuring that the tissue structure and molecular composition are maintained for accurate
histological analysis.
Types of Fixatives
Fixatives can be broadly classified into two categories:
chemical and
physical fixatives.
Chemical Fixatives: These are the most commonly used fixatives and include aldehydes (e.g., formaldehyde), alcohols (e.g., ethanol), and oxidizing agents (e.g., osmium tetroxide). Each type has specific applications depending on the tissue and the intended analysis.
Physical Fixatives: These involve methods like
freeze-fixation, where tissues are rapidly frozen to preserve their state. This method is less common but useful for certain applications, such as electron microscopy.
Common Fixatives and Their Properties
Several fixatives are routinely used in histology, each with unique properties: Formaldehyde: Also known as
formalin when in aqueous solution, it is the most widely used fixative. It penetrates tissues well and preserves proteins by forming cross-links. Formalin-fixed tissues are suitable for a wide range of staining techniques.
Glutaraldehyde: This fixative is often used for
electron microscopy as it provides excellent preservation of ultrastructure. However, it penetrates tissues more slowly than formaldehyde.
Osmium Tetroxide: Primarily used for electron microscopy, it preserves lipids and provides excellent membrane contrast. However, it is highly toxic and requires careful handling.
Alcohols: Ethanol and methanol are often used for cytological smears and to fix tissues for RNA and DNA analysis. They work by precipitating proteins and disrupting cell membranes.
The Fixation Process
The fixation process involves several critical steps to ensure optimal preservation: Tissue Collection: Tissues must be harvested and fixed as quickly as possible to prevent degradation. The size and thickness of the tissue also influence penetration and fixation quality.
Fixative Choice: The selection of an appropriate fixative depends on the tissue type, the analysis to be performed, and the downstream processing techniques.
Fixation Time: The duration of fixation is crucial. Under-fixation can lead to poor preservation, while over-fixation can cause excessive cross-linking, making tissues hard and brittle.
Fixative Volume: An adequate volume of fixative (usually 10-20 times the tissue volume) is necessary to ensure complete penetration and preservation.
Temperature: Fixation is typically carried out at room temperature, but some protocols may require lower temperatures to preserve certain molecules.
Potential Issues and Solutions
Several issues can arise during fixation, but they can often be mitigated with careful planning and technique: Incomplete Fixation: Ensure proper tissue size, fixative volume, and fixation time to avoid areas of unfixed tissue, which can lead to artifacts.
Over-Fixation: Excessive fixation can make tissues hard and difficult to section. Monitoring fixation time and using appropriate fixatives can help prevent this.
Precipitation of Fixative: Some fixatives can precipitate, especially at low temperatures. Ensure proper storage and handling of fixative solutions.
Conclusion
Fixation is a complex but essential process in histology that ensures the preservation of tissue morphology and molecular integrity. By understanding the principles of fixation and carefully selecting and applying fixatives, researchers and pathologists can achieve reliable and accurate histological analyses.
