Fixation is a critical step in histology that involves preserving biological tissues from decay and maintaining their structural integrity. This process prevents autolysis and bacterial decomposition, ensuring that the tissues retain their microscopic anatomy. Fixation is commonly achieved using chemical agents called
fixatives.
The primary goal of fixation is to stabilize the tissues in a life-like state. This is crucial for accurate microscopic examination and diagnosis. Proper fixation preserves cellular and subcellular structures, making them visible under a microscope. It also enhances the staining properties of tissues, allowing for better differentiation of various cellular components.
Several fixatives are used in histology, each with specific properties suitable for different tissue types and purposes. Common fixatives include:
Formaldehyde: Widely used due to its ability to preserve a broad range of tissues.
Glutaraldehyde: Effective for electron microscopy due to its ability to cross-link proteins rapidly.
Alcohol (Ethanol or Methanol): Used for cytological smears and quick fixation.
Bouin's Solution: Excellent for soft and delicate tissues, enhancing color contrast.
Embedding is the process of enclosing fixed tissues in a solid medium to facilitate sectioning. After fixation, tissues are typically infiltrated with a medium such as paraffin wax or resin, which provides the necessary support for thin sectioning. Embedding ensures that tissues retain their orientation and structure during the
sectioning process.
Embedding is essential for obtaining thin, uniform tissue sections that can be mounted on slides for microscopic examination. The embedding medium provides rigidity, preventing the tissue from collapsing or distorting during sectioning. This step is crucial for achieving precise and consistent histological analysis.
Common embedding media include:
Paraffin Wax: The most commonly used medium due to its ease of use and compatibility with various staining techniques.
Resin: Used for high-resolution microscopy, especially in electron microscopy.
Agarose: Suitable for specific applications, such as maintaining the orientation of small or delicate tissues.
The embedding process involves several steps:
Dehydration: Removal of water from the tissue using a series of increasing concentrations of alcohol.
Clearing: Replacement of alcohol with a clearing agent like xylene, which is miscible with both alcohol and the embedding medium.
Infiltration: Penetration of the tissue with the embedding medium, usually at an elevated temperature for paraffin.
Embedding: Placement of the tissue in a mold filled with embedding medium, followed by cooling to solidify the medium.
Both fixation and embedding present certain challenges:
Over-fixation or under-fixation can lead to poor preservation of tissue morphology.
Inadequate infiltration with the embedding medium can result in incomplete support, affecting section quality.
Compatibility of the fixative and embedding medium with subsequent
staining techniques must be considered.
Conclusion
Fixation and embedding are fundamental steps in histology that ensure the preservation and preparation of tissues for microscopic examination. Understanding the principles and techniques involved in these processes is essential for obtaining accurate and reliable histological results. Properly fixed and embedded tissues provide a clear and detailed view of cellular structures, enabling effective diagnosis and research.
