Optimal FFPE Tissue Fixation, Processing, and Embedding: A Comprehensive Guide

September 4, 2024

Optimal fixation, processing, and embedding of Formalin-Fixed, Paraffin-Embedded (FFPE) tissue are crucial steps in histological examination. These initial stages ensure high-quality staining, imaging, and quantitative analysis of tissue morphology and biomarker localization. This guide provides an overview of common fixation, processing, and embedding methods for FFPE rodent tissue.

Fixation for FFPE Tissue

Proper tissue fixation preserves the tissue architecture, including cells, cellular components (cytoplasm, nuclei, organelles), extracellular material, and molecular components (proteins, DNA, mRNA). Various tissue fixatives are available, each with specific advantages and disadvantages depending on the application. Timing is also key. The cold ischemia time, time from removal of tissue until placement in fixative, should not exceed 1 hour.

Common Fixative: 10% Neutral Buffered Formalin (10% NBF)

10% NBF is the most widely used fixative in routine diagnostic pathology due to its ability to preserve tissue effectively. Key factors for optimal results include:

• Specimen Thickness: Trim tissue to 2-3 mm before placing it in fixative.
• Volume Ratio: Use a minimum of one part tissue to 20 parts fixative. Ensure the fixative is free of blood.
• Fixation Time: Depending on sample size, tissues are fixed for 6 to 72 hours at room temperature before processing into paraffin wax.
• Storage: Store tissues in 70% ethanol post-fixation, though this is not recommended for brain or neurological samples.

For tissues like eyes, lungs, intestines, and bones that may require different fixatives or special processing, contact our expert team for guidance on the appropriate tissue fixation techniques.

Processing for and Embedding of FFPE Tissue

Fixed tissues are typically processed using automated tissue processors and embedded in paraffin wax. This process involves immersing samples in a series of solutions, including alcohols, xylene, and paraffin wax. Customization of each step—time, temperature, and pressure/vacuum (P/V)—ensures optimal solution penetration.

Our histologists have developed specific tissue processing schedules tailored to different tissue types, ensuring high-quality outcomes. Understanding the steps of tissue processing is essential for maintaining the integrity of FFPE samples.

High-quality embedding requires consistent orientation of all samples of the same tissue type. While samples can be embedded in various orientations upon request, we typically follow the Registry of Industrial Toxicology Animal (RITA) and North American Control Animal Database (NACAD) Guidelines for rodent tissue embedding.

If your study requires a different tissue embedding orientation, please specify in advance, and we will accommodate your needs.

Keeping FFPE on top of mind

Optimal fixation, processing, and embedding are critical steps for examining tissue morphology and biomarker localization in FFPE tissue samples. Our expert team can assist you with all aspects of formalin-fixed paraffin-embedded tissue preparation. Contact us if you have any questions, our team is here to ensure your histology workflow is optimized for the best results.