What is Tissue Pre-Treatment in Histology?
Tissue pre-treatment in histology refers to the initial steps taken to prepare biological tissues for subsequent processing, analysis, and
microscopic observation. These steps are crucial to preserve the tissue's cellular structure, preventing degradation and ensuring that the tissue is in the best possible condition for staining, sectioning, and examination.
Fixation
Fixation is the primary step in tissue pre-treatment. It involves immersing tissues in a
fixative solution to stabilize and preserve cellular components. Common fixatives include
formalin,
glutaraldehyde, and
ethanol. The choice of fixative depends on the type of tissue and the intended downstream applications.
Dehydration
Following fixation, tissues must be dehydrated to remove water content. This is typically achieved by passing tissues through a series of increasing concentrations of ethanol. Dehydration is essential for subsequent embedding in non-aqueous media such as paraffin.
Clearing
Clearing replaces the ethanol in tissues with a substance that is miscible with both ethanol and the embedding medium.
Xylene is a widely used clearing agent, although alternatives like
toluene and
cedarwood oil can also be used. Proper clearing ensures that tissues are transparent and ready for embedding.
Embedding
Embedding involves infiltrating the cleared tissues with a medium such as
paraffin wax or resin, which solidifies to provide support and structure for sectioning. Choosing the appropriate embedding medium is critical for maintaining tissue morphology and facilitating thin sectioning.
Sectioning
Once embedded, tissues are sectioned into thin slices using a
microtome. These sections are typically 3-5 micrometers thick and are mounted onto glass slides for staining and examination. Proper sectioning ensures uniform thickness and minimal artifact formation.
Over-Fixation or Under-Fixation
Over-fixation can lead to tissue hardening, while under-fixation can result in poor preservation. To avoid these issues, it is important to use the correct concentration of fixative and adhere to the recommended fixation time for each tissue type.
Incomplete Dehydration
Incomplete dehydration can cause poor infiltration of the embedding medium, leading to artifacts in tissue sections. Ensuring thorough dehydration by extending the time in higher ethanol concentrations can mitigate this issue.
Inadequate Clearing
Inadequate clearing can result in opaque tissues that are difficult to section. Using fresh clearing agents and ensuring sufficient clearing time can help achieve optimal tissue transparency.
Embedding Artifacts
Embedding artifacts such as air bubbles or improper orientation can compromise the quality of tissue sections. Careful handling and proper technique during embedding can minimize these artifacts.
Conclusion
Tissue pre-treatment is a critical aspect of histology that ensures the preservation and quality of tissue samples for microscopic analysis. By understanding and properly executing the steps of fixation, dehydration, clearing, embedding, and sectioning, histologists can achieve high-quality tissue preparations, leading to accurate and reliable diagnostic results.
