What is Post Fixation?
Post fixation is a crucial step in the preparation of tissue samples for microscopic examination in
histology. It involves treating the fixed tissue with additional reagents to enhance preservation, contrast, and staining properties. This step follows the initial fixation process, where tissues are preserved to prevent decay and maintain structural integrity.
Why is Post Fixation Necessary?
The primary purpose of post fixation is to improve the quality of the tissue sample. Initial fixation can sometimes leave tissues inadequately preserved or poorly stained. Post fixation helps by:
- Enhancing contrast in the tissue sections.
- Improving the penetration of
stains.
- Stabilizing cellular components for better visualization.
- Reducing background staining and artifacts.
Common Reagents Used in Post Fixation
Several reagents are commonly employed in post fixation to achieve the desired outcomes:
-
Osmium tetroxide: Often used for electron microscopy to provide excellent contrast and preserve lipids.
-
Ethanol: Used to dehydrate tissues and improve stain uptake.
-
Acetic acid: Helps in fixing nucleoproteins and enhancing nuclear staining.
-
Mercuric chloride: Enhances the preservation of fine cellular structures.
How is Post Fixation Performed?
The process of post fixation involves several steps:
1.
Initial Fixation: The tissue is first fixed using a primary fixative like formaldehyde.
2.
Rinsing: The fixed tissue is rinsed to remove excess fixative.
3.
Post Fixation Treatment: The tissue is then treated with post-fixative reagents, which may vary based on the intended analysis.
4.
Dehydration: This step involves passing the tissue through a series of graded alcohols to remove water.
5.
Clearing and Embedding: The tissue is cleared using a solvent like xylene and then embedded in a suitable medium like paraffin for sectioning.
Challenges in Post Fixation
While post fixation is beneficial, it presents certain challenges:
- Over-fixation: Prolonged exposure to post-fixative reagents can lead to over-fixation, causing tissue hardening and difficulty in sectioning.
- Compatibility: Not all post-fixative reagents are compatible with every type of tissue or stain, requiring careful selection.
- Artifact Formation: Improper post fixation can introduce artifacts that may interfere with accurate interpretation.Applications of Post Fixation
Post fixation is widely used in various applications within histology:
- Electron Microscopy: Post fixation with osmium tetroxide is essential for the detailed visualization of cellular ultrastructure.
- Immunohistochemistry: Enhances the binding of antibodies to antigens, improving the specificity and sensitivity of staining.
- Histochemical Staining: Improves the precision and clarity of stains used to identify specific molecules within tissues.Best Practices for Post Fixation
To achieve optimal results, consider the following best practices:
- Standardization: Follow standardized protocols to ensure consistency and reproducibility.
- Timing: Carefully monitor the duration of post fixation to avoid over-fixation.
- Selection of Reagents: Choose post-fixative reagents based on the specific requirements of the tissue and the intended analysis.
- Documentation: Keep detailed records of all steps and reagents used to facilitate troubleshooting and reproducibility.Conclusion
Post fixation is a vital step in the histological preparation of tissue samples, enhancing their preservation and staining properties. By understanding the principles, reagents, and best practices involved, histologists can significantly improve the quality and reliability of their microscopic analyses.
