What is Tissue Fixation?
Tissue fixation is a crucial step in the preparation of biological tissues for
microscopic examination. The primary purpose of fixation is to preserve the tissue in a life-like state, preventing
autolysis (self-digestion) and
putrefaction (decomposition by bacteria and fungi). This process stabilizes the tissue structure, allowing for detailed analysis and accurate diagnosis.
Types of Fixatives
There are various types of fixatives used in histology, each with specific advantages and applications. The most common categories include: Formaldehyde (commonly used as formalin): It is a widely used fixative that preserves a broad range of tissues and is compatible with many staining techniques.
Glutaraldehyde: Frequently used for electron microscopy due to its ability to preserve fine cellular details.
Alcohol (such as ethanol and methanol): Often used for cytological preparations and DNA analysis.
Mercurials (such as B-5 fixative): Utilized for hematopoietic and lymphoid tissues.
Picrates (such as Bouin's solution): Excellent for preserving soft and delicate tissues.
Choosing the Right Fixative
The choice of fixative depends on several factors, including the type of tissue, the intended analysis, and the desired staining techniques. For example, formalin is suitable for routine histology, while glutaraldehyde is preferred for electron microscopy. It is crucial to match the fixative with the specific requirements of the study to achieve optimal results.Fixation Protocol
The fixation process typically involves the following steps: Tissue Collection: Carefully excise the tissue to avoid mechanical damage and place it immediately in the fixative to prevent autolysis.
Fixative Volume: Use an adequate volume of fixative, usually 10-20 times the volume of the tissue, to ensure complete penetration.
Fixation Time: The duration of fixation varies depending on the fixative and tissue type. Formalin fixation usually requires 6-24 hours, while glutaraldehyde fixation for electron microscopy may take 1-2 hours.
Temperature: Fixation is typically performed at room temperature, although some protocols may require refrigeration or heating.
Common Fixation Issues and Solutions
Improper fixation can lead to various issues, including poor tissue preservation,
shrinkage, and
hardening. Here are some common problems and their solutions:
Incomplete Fixation: Ensure the tissue is thinly sliced (usually 3-4 mm) to facilitate complete penetration of the fixative.
Over-fixation: Follow the recommended fixation time to avoid over-hardening the tissue, which can make sectioning difficult.
Fixative Precipitation: Use fresh fixative and avoid prolonged storage, as some fixatives can precipitate over time.
Post-Fixation Processing
After fixation, tissues undergo further processing steps, including
dehydration,
clearing, and
embedding. Proper fixation is critical for the success of these subsequent steps, as it influences the quality of tissue sections and the effectiveness of staining protocols.
Conclusion
Proper tissue fixation is a cornerstone of histological practice. By preserving tissue morphology and biochemistry, fixation enables accurate microscopic examination and diagnosis. Understanding the principles of fixation, choosing appropriate fixatives, and following meticulous protocols are essential for achieving high-quality histological preparations.
