What is the critical step in the deparaffinization process?

In the deparaffinization process, which is crucial for preparing tissue samples for microscopic examination, using xylene two times is recognized as the critical step for reducing contamination. Xylene acts as a solvent that dissolves the paraffin wax embedded in tissue samples, allowing for better penetration of staining solutions in subsequent steps.

Using xylene two times is specifically emphasized because it ensures thorough removal of the paraffin. The first treatment often helps to remove the bulk of the paraffin, while the second treatment ensures that any residual wax is effectively addressed. This two-step method not only enhances the clarity and definition of stained tissue sections but also minimizes the risk of contamination that could arise from inadequate paraffin removal.

In contrast, using xylene three times might be considered excessive for routine procedures, while heating paraffin to high temperatures is not applicable to the deparaffinization phase, as it can lead to altered tissue morphology instead of aiding in paraffin removal. Additionally, removing stains prior to embedding is a step that occurs at a different stage of sample preparation and does not pertain directly to the deparaffinization process itself. Thus, the chosen answer highlights the importance of careful and sufficient solvent use to achieve optimal results when preparing tissue samples for analysis