What is Fixation in Histology?
Fixation is a critical step in histology that involves preserving biological tissues from decay and preparing them for subsequent examination under a microscope. The process stabilizes the tissue structure and prevents enzymatic degradation and microbial growth.
Why is Fixation Important?
Proper fixation is crucial for maintaining the
morphological integrity and biochemical composition of tissues. It allows for accurate histological analysis and diagnosis. Inadequate fixation can lead to tissue shrinkage, distortion, and loss of cellular details, compromising the quality of the histological examination.
What Fixatives are Commonly Used?
A variety of fixatives are used in histology, each with specific properties and applications. Some of the most common fixatives include:
Formaldehyde (commonly used as formalin): A universal fixative that preserves a wide range of tissues.
Glutaraldehyde: Often used for electron microscopy due to its ability to cross-link proteins more effectively.
Alcohol-based fixatives: Used for preserving cytological specimens and for some immunohistochemical applications.
Zinc-based fixatives: Provide superior preservation of antigenicity for immunohistochemistry.
How to Optimize Fixation Time?
Fixation time is critical and varies depending on the type and size of the tissue. Over-fixation can cause excessive cross-linking, leading to hardening and difficulty in sectioning, while under-fixation can result in incomplete preservation. Here are some guidelines to optimize fixation time:
Small biopsies generally require 4-6 hours of fixation.
Larger tissue samples may need 24-48 hours.
For electron microscopy, shorter fixation times are often sufficient due to the small size of specimens.
pH: Most fixatives work optimally at a pH range of 7.2-7.4. Deviations from this range can affect the protein structure and tissue morphology.
Temperature: Fixation is usually carried out at room temperature (20-25°C). Higher temperatures can accelerate fixation but may also increase the risk of tissue artifacts.
Tissue thickness: Thinner sections (2-4 mm) allow for better penetration.
Fixative volume: A volume ratio of 10:1 (fixative:tissue) is generally recommended to ensure adequate coverage.
Agitation: Gentle agitation of the fixative can enhance penetration.
Shrinkage: Can be minimized by using appropriate fixative concentrations and fixation times.
Formalin pigment: Avoided by using buffered formalin solutions.
Cross-linking artifacts: Minimized by choosing the right fixative and optimizing fixation conditions.
Can Fixation Affect Subsequent Staining?
Yes, fixation can impact subsequent staining procedures. For example, certain fixatives may mask antigenicity, affecting
immunohistochemical staining. It is essential to select a fixative that is compatible with the intended downstream applications. Pre-treatments such as antigen retrieval can sometimes be employed to overcome fixation-induced masking.
How to Store Fixed Tissues?
After fixation, tissues should be stored properly to maintain their integrity. Fixed tissues can be stored in the same fixative or transferred to a suitable storage solution. Long-term storage should be at 4°C to prevent degradation.
Conclusion
Optimizing fixation in histology requires a careful balance of various factors, including fixative type, fixation time, pH, temperature, and tissue handling. By understanding and controlling these variables, high-quality histological preparations can be achieved, facilitating accurate microscopic analysis and diagnosis.
