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.
