Tissue processing is the technique by which fixed tissues are prepared to be embedded in a solid medium, typically paraffin wax, for microscopic examination. This process transforms soft, fragile biological tissues into durable specimens that can be thinly sliced and stained.
The Three Core Steps of Tissue Processing
The process fundamentally involves three main sequential steps:
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Dehydration: This step removes water from the tissue. Tissues are immersed in a series of increasing concentrations of alcohol (e.g., ethanol) to gradually replace the water content. For example, tissues might be processed through 70%, 80%, 90%, 95%, and finally 100% ethanol solutions. The goal is to prevent distortion during the subsequent infiltration step.
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Clearing: Following dehydration, the alcohol must be replaced with a solvent that is miscible (mixable) with both alcohol and the embedding medium (e.g., paraffin). Common clearing agents include xylene, toluene, and limonene-based reagents. Clearing agents also make the tissue more transparent.
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Infiltration: This is the process of replacing the clearing agent with the embedding medium. Molten paraffin wax is the most common embedding medium. The tissue is immersed in several changes of hot paraffin wax (typically around 55-60°C) under vacuum to ensure complete penetration of the wax into the tissue. This supports the tissue structure, allowing for thin sectioning.
Why is Tissue Processing Necessary?
Without tissue processing, it would be impossible to create the thin, uniform sections required for microscopic examination. The process hardens the tissue, supporting its cellular structure and enabling microtomy (thin sectioning).
Summary
In summary, tissue processing is a crucial series of steps necessary to prepare biological tissues for microscopic examination. This involves dehydration, clearing, and infiltration, resulting in a tissue sample embedded in a solid medium, typically paraffin wax, ready for sectioning and staining.
