What is Deparaffinization?
In the field of
histology, deparaffinization refers to the process of removing paraffin wax from tissue sections that have been embedded in paraffin blocks. This step is crucial for preparing tissue sections for subsequent staining and microscopic examination. Paraffin wax is used to preserve and support tissue morphology during sectioning, but it needs to be removed for the tissue sections to be properly stained.
Why is Deparaffinization Important?
Deparaffinization is essential because paraffin wax can interfere with the penetration of staining reagents, leading to suboptimal staining results. Proper deparaffinization ensures that the tissue sections are free of wax, allowing stains to bind specifically to cellular and tissue components. This is particularly important for diagnostic purposes, where accurate identification of cellular structures is critical.
Xylene Treatment: The tissue sections are first immersed in
xylene or a xylene substitute to dissolve the paraffin wax. This step is usually repeated several times to ensure complete removal of the wax.
Graded Alcohol Series: After xylene treatment, the sections are passed through a series of decreasing concentrations of alcohol (usually ethanol). This step helps to remove any remaining xylene and prepares the tissue for hydration.
What is Hydration?
Hydration is the process of reintroducing water into the tissue sections after they have been deparaffinized. This step is crucial for preparing the tissue sections for staining, as most histological stains are water-based. Proper hydration ensures optimal staining and visualization of tissue components.
How is Hydration Performed?
Hydration involves passing the tissue sections through a series of decreasing concentrations of alcohol, followed by immersion in water. Here are the main steps:
Alcohol Series: The sections are passed through a series of decreasing concentrations of alcohol (ethanol), usually starting from 100% ethanol and moving to 95%, 70%, and finally to water. This step gradually reintroduces water into the tissue.
Water Rinse: The final step involves rinsing the tissue sections in distilled or deionized water to remove any remaining alcohol. This step prepares the tissue for staining.
Incomplete Deparaffinization: Insufficient deparaffinization can result in residual wax, leading to poor staining. Ensuring thorough
deparaffinization with multiple xylene treatments can help mitigate this issue.
Overhydration: Prolonged exposure to water can lead to tissue swelling and damage. Careful monitoring of the hydration steps can prevent overhydration.
Alcohol Evaporation: Alcohol can evaporate quickly, especially in open containers, leading to inconsistent concentrations. Using tightly sealed containers and monitoring alcohol levels can help maintain consistency.
Citrus-Based Solvents: These solvents are less toxic and have been shown to effectively remove paraffin wax without compromising tissue integrity.
Aliphatic Hydrocarbons: These are less toxic than xylene and have similar deparaffinizing properties.
Conclusion
Deparaffinization and hydration are critical steps in the preparation of tissue sections for histological examination. Proper execution of these steps ensures optimal staining and accurate visualization of tissue components. By understanding the importance and methods of deparaffinization and hydration, histologists can achieve high-quality results in their diagnostic and research endeavors.
