What is Fixing in Histology?
In
histology, fixing refers to the process of preserving biological tissues to prevent autolysis and decay. This is a crucial step in preparing samples for
microscopy and other analytical techniques. Fixation stabilizes the tissue's structure and maintains the distribution of cellular components, making it easier to study under a microscope.
Why is Fixing Important?
Fixing is essential because it preserves the tissue in a state as close to life-like as possible. This is critical for accurate
histological examination and diagnosis. Without proper fixing, tissues can degrade, leading to loss of structure and function, which would compromise the results of the analysis.
What are the Common Fixatives?
Several fixatives are commonly used in histology, each with unique properties suited for different types of tissues and analyses:
Formaldehyde: Often used as a 10% neutral buffered formalin solution, it cross-links proteins, preserving tissue morphology.
Glutaraldehyde: Provides better cross-linking than formaldehyde, making it ideal for electron microscopy.
Alcohols: Ethanol and methanol are used for fixing cytological smears and nucleic acid preservation.
Osmium tetroxide: Used primarily in electron microscopy for its excellent lipid preservation.
Bouin's solution: A combination of picric acid, formaldehyde, and acetic acid, used for preserving soft and delicate tissues.
How is Fixing Performed?
Fixing is typically performed by immersing the tissue in the fixative solution. The duration of fixation can vary depending on the type of tissue and the fixative used. For formaldehyde, fixation might take from a few hours to overnight, whereas glutaraldehyde fixation for electron microscopy is usually shorter, lasting a few hours.
pH: Most fixatives work best at a neutral pH (around 7.0).
Temperature: Fixation is generally performed at room temperature, although some rapid fixations are done at lower temperatures to slow down enzymatic activity.
Concentration: The concentration of the fixative affects the rate and extent of fixation. Higher concentrations may fix tissues faster but can also lead to over-fixation and tissue hardening.
Time: The duration of fixation must be optimized; prolonged fixation can lead to excessive hardening and shrinkage, while insufficient fixation can result in poor preservation.
Penetration: Fixatives must penetrate tissues adequately. Larger tissues may require slicing to ensure proper fixation throughout the sample.
Artifact Formation: Improper fixation can lead to artifacts, which are structural abnormalities not present in the living tissue.
Loss of Antigenicity: Some fixatives can mask or destroy antigens, making subsequent
immunohistochemical staining problematic.
Tissue Deformation: Over-fixation can cause tissue hardening and shrinkage, altering the natural morphology.
Conclusion
Fixing is a pivotal step in histological processing and ensures that tissues are preserved in a state suitable for detailed examination. Understanding the types of fixatives, their applications, and the factors that influence fixation can significantly impact the quality of histological analyses.
