Optimal fixation, processing, and embedding are critical first steps to allow the histological examination of tissue morphology and biomarker localization in tissue samples. Care at these initial stages leads to higher quality staining, imaging, and molecular analysis at later phases of the histology workflow. This article is a brief overview of common fixation, processing, and embedding schemes for FFPE rodent tissue.
Optimal fixation of histological samples is essential for the preservation of tissue architecture including cells, cellular components (i.e., cytoplasm, nuclei, organelles), extracellular material, and molecular components (i.e., proteins, DNA, mRNA). There are many different fixatives available, and each has their advantages and disadvantages depending on application. You can read more about different tissue fixatives using Google’s Talk to Books search.
The most common fixative used in routine diagnostic pathology is 10% Neutral Buffered Formalin (10% NBF). This fixative typically results in good tissue preservation; however, factors such as fixation time, temperature, pH, and specimen thickness must be taken into account in order to achieve the best results.
Specimens: tissue should be trimmed to a thickness of 3 – 5 mm before being placed in fixative.
Volume: at least one part tissue to 20 parts fixative should be used. Fixative should be free of blood.
Time: tissues are commonly fixed for 24 hours at room temperature prior to processing into paraffin wax.
Storage: tissues may be stored in 70% ethanol after fixation. This is not recommended for brain or other neurological samples.
Tissues such as eyes, lungs, intestines, and bones may require different fixatives and/or special processing, please contact Reveal Biosciences for more information.
Fixed tissues are typically processed into paraffin wax using automated tissue processors. Automated tissue processing involves immersing samples in a defined series of solutions including alcohols, xylene, and paraffin wax. Each step can be customized by controlling time, temperature, and pressure / vacuum (P/V) to ensure good solution penetration into the samples. Our histologists have developed a number of tissue processing schedules specifically designed for different tissue types.
High quality embedding involves the consistent orientation of all samples of the same tissue type. Samples can be embedded into blocks in a variety of orientations on request; however, we typically follow the Registry of Industrial Toxicology Animal (RITA) and North American Control Animal Database (NACAD) Guidelines for rodent tissue embedding. Please be sure to specify in advance if your study requires a different tissue embedding orientation and we’ll be happy to accommodate your request.
In summary, optimal fixation, processing, and embedding are critical first steps to allow the examination of tissue morphology and biomarker localization in tissue samples. Please contact us with any questions or to discuss your study needs and we’ll be happy to help.