The Deparaffinization & Rehydration of FFPE Tissue Samples

When it comes to analyzing formalin-fixed, paraffin-embedded (FFPE) tissue samples, one of the critical preparatory steps is deparaffinization. FFPE samples are commonly used in research, histology, and diagnostic pathology because the paraffin wax preserves the tissue structure. However, to perform downstream analyses like staining, immunohistochemistry (IHC), or molecular studies, the paraffin must be removed, and the tissue must be rehydrated. This process, known as deparaffinization and rehydration, is vital for researchers and pathologists who rely on accurate tissue sample analysis.

Let’s explore the purpose, protocol, and steps involved in deparaffinization and rehydration, offering insights on how to achieve the best results with your FFPE samples.

What is Deparaffinization?

Deparaffinization refers to the process of removing paraffin wax from FFPE tissue sections. Paraffin wax is used to preserve tissue architecture during embedding, but before any molecular or histological analysis can be performed, the wax must be removed. This process is essential for subsequent staining procedures or molecular assays that require access to the proteins, RNA, and DNA within the tissue sections.

The purpose of deparaffinization is to return the tissue to its original state before it was embedded in wax, allowing researchers and clinicians to analyze it for pathological or diagnostic purposes.

Why is Deparaffinization Important?

Proper deparaffinization is crucial to obtaining reliable, reproducible results. The incomplete or improper removal of paraffin can hinder the efficiency of downstream processes such as staining, DNA extraction, or protein isolation. Tissue that still contains paraffin can lead to false negatives in assessments or poor visualization to ensure accurate results.

Before starting the deparaffinization process, tissue sections are usually mounted onto glass slides. These tissue sections should be handled with care to ensure they are securely attached and free from any debris. Typically, sections are cut between 4 and 6 microns thick and adhered to the slides using a section adhesive for microscopic examination.

Deparaffinization Protocol: How to Remove Paraffin from FFPE Samples

Xylene is the most widely used solvent in the deparaffinization of tissue sections. It dissolves the paraffin wax, enabling it to be removed from the tissue. For effective deparaffinization, the slides are typically immersed in xylene or a xylene substitution for 2–3 cycles, with each immersion lasting between 5 and 10 minutes. Xylene is highly effective but also toxic, so appropriate safety measures—such as working in a well-ventilated space or using xylene substitutes—should be followed.

The first immersion into the xylene bath begins dissolving the paraffin. After 5–10 minutes, the slides are transferred to the next bath. A second xylene bath ensures any remaining paraffin is fully dissolved, further improving the quality of deparaffinization. Researchers may choose to perform a third xylene bath for thicker or older sections.

FFPE Deparaffinization and Rehydration

After the paraffin is removed, the tissue needs to be rehydrated to restore its original state. This is done by gradually decreasing the concentration of ethanol, which is critical to prevent tissue damage. This step also helps eliminate any residual xylene that may still be present in the sample.

The rehydration process typically follows these 4 steps:

100% Ethanol: The slides are immersed in absolute ethanol for 5 minutes to remove any remaining xylene.
95% Ethanol: The slides are transferred to a solution of 95% ethanol for another 5 minutes. This step starts the gradual rehydration process.
70% Ethanol: The next step involves placing the slides in 70% ethanol, which further hydrates the tissue.
50% Ethanol: The slides are then moved to 50% ethanol to ensure full hydration.

By the end of this step, the tissue has been completely rehydrated and is ready for further analysis.

Best Practices for Proper Deparaffinization

The success of the deparaffinization process depends on carefully following the protocol and optimizing the conditions for each type of tissue. Here are a few tips on how to deparaffinize FFPE tissue slides efficiently:

1. Ensure Adequate Immersion Time

The slides should remain in xylene long enough to dissolve the paraffin fully. However, be careful not to leave them in too long, as this can damage delicate tissues.

2. Monitor Ethanol Gradients

The transition from xylene to ethanol and through the ethanol series must be done gradually to avoid dehydration or overhydration of the tissue. Skipping steps in the gradient can lead to poor results.

3. Optimize for Tissue Thickness

Thicker tissue sections may require longer exposure to xylene or more baths to completely remove the paraffin.

Challenges in Deparaffinization and Rehydration

One of the main challenges in deparaffinization is ensuring that all paraffin is removed from the sample without damaging the tissue. Incomplete deparaffinization can result in improper staining or molecular analysis, leading to false negatives or weak signals. In contrast, overexposure to solvents such as xylene can cause the tissue to become more brittle or distorted.

Additionally, working with different tissue types can present unique challenges. Some tissues may require longer xylene immersion times or greater rehydration protocols. Understanding the characteristics of your sample is essential to modifying your deparaffinization protocol accordingly.

Order Samples You Can Trust From Superior BioDiagnostics

Deparaffinization is a critical step in preparing FFPE tissue sections for accurate analysis, from staining to molecular research. Ensuring proper deparaffinization and rehydration can make all the difference in obtaining reliable results. If you’re working with FFPE samples and want to guarantee the highest quality preparation, Superior BioDiagnostics is here to help.

With our expert deparaffinization protocol and tissue handling services, we ensure your orders are in optimal condition for your research or diagnostic needs. Our trusted Biobank has all the samples you need for your next discovery, including breast, brain, lung, skin, cervical, colonoscopy, endoscopy, endometrial, epithelial, and muscular tissue. Don’t leave the quality of your tissue samples to chance–contact us today to learn how our specialized solutions can support your lab or clinical work, and let us handle the intricacies of tissue preparation with precision and care!