What is Fixation in Histology?
Fixation is a crucial step in histological processes that involves preserving biological tissues from decay and preparing them for further examination. This procedure stabilizes the tissue's structure, maintains cellular integrity, and prevents enzymatic degradation and autolysis.
Why is Fixation Important?
Fixation is essential because it preserves tissue morphology and molecular composition, which is vital for accurate diagnosis and research. Without proper fixation, the tissue could deteriorate, leading to inaccurate or misleading results.
Types of Fixatives
There are two main categories of fixatives: chemical and physical. Chemical Fixatives
Chemical fixatives are the most commonly used and can be further divided into:
Formaldehyde: A widely used fixative that forms cross-links between proteins, preserving structure. Formalin, a 10% solution of formaldehyde, is commonly used in labs.
Glutaraldehyde: Another aldehyde fixative, often used for electron microscopy due to its ability to preserve ultrastructure.
Alcohols: Ethanol and methanol are dehydrating agents that precipitate proteins and are used for cytological smears.
Acetic Acid: Used in combination with other fixatives to preserve nucleic acids.
Osmium Tetroxide: Primarily used in electron microscopy for its ability to fix lipids and provide contrast.
Physical Fixatives
Physical methods include freezing and microwave irradiation. While not as common as chemical fixation, these methods are used in specific scenarios where rapid fixation is required.
Tissue Type: Different tissues may require different fixatives for optimal preservation. For example, brain tissue often requires a fixative that preserves the soft structure, like formalin.
Purpose of Study: The end goal, whether it be light microscopy, electron microscopy, or molecular analysis, influences the choice of fixative.
Time and Resources: Some fixatives require longer fixation times and more complex handling than others.
Fixation Protocols
General Fixation Protocol
A typical fixation process involves several steps: Sample Collection: Fresh tissue samples are collected and immediately immersed in fixative to prevent degradation.
Fixative Preparation: The fixative is prepared according to the required concentration and pH.
Immersion: The tissue is immersed in the fixative, ensuring complete coverage. The volume of fixative should be at least 10 times the volume of the tissue.
Incubation: The tissue is incubated in the fixative for an appropriate amount of time, typically ranging from a few hours to overnight, depending on the tissue type and fixative.
Post-Fixation: In some cases, tissues are transferred to a secondary fixative or a buffer solution to wash out excess fixative and prepare the tissue for subsequent processing.
Specific Fixation Protocols
Formalin Fixation
1. Collect the tissue sample and immediately immerse it in 10% neutral buffered formalin.
2. Incubate the tissue at room temperature for 24 hours.
3. Rinse the tissue with water to remove any formalin residues.
Glutaraldehyde Fixation for Electron Microscopy
1. Collect small tissue pieces (1-2 mm) and immerse them in 2.5% glutaraldehyde solution.
2. Fix the tissue at 4°C for 2-4 hours.
3. Rinse the tissue with a buffer solution (e.g., phosphate buffer) to remove excess glutaraldehyde.
Common Problems and Solutions
Incomplete Fixation
Incomplete fixation can lead to poor tissue preservation and artifacts. To avoid this, ensure that the tissue is adequately immersed in the fixative and use a sufficient volume of fixative relative to the tissue size. Over-Fixation
Over-fixation can cause tissue hardening and difficulty in sectioning. To prevent this, adhere to the recommended fixation times for each fixative.
Artifacts
Artifacts can arise from improper fixation techniques. Ensure that fixatives are fresh and properly prepared, and that tissues are handled gently to minimize mechanical damage.
Conclusion
Fixation is a vital step in histology that significantly impacts the quality and reliability of tissue analysis. By understanding the different types of fixatives, selecting the appropriate one for the tissue and study purpose, and following proper fixation protocols, histologists can ensure the preservation of tissue morphology and molecular integrity for accurate examination and diagnosis.
Further Reading
For more detailed information on fixation techniques and protocols, consider exploring additional resources and literature on histological methods and tissue preservation.
