Formalin-fixed paraffin-embedded (FFPE) tissue samples are preserved using formalin and then embedded in paraffin wax and are commonly used as control samples in medical diagnostics. FFPE control tissue samples are great for experimental tests. The wax allows researchers to examine multiple extremely thin and fragile sections from a single biopsy.

According to the National Library of Medicine, “Pathology departments routinely archive vast numbers of FFPE blocks as compared to frozen tissues. This largely untapped resource represents an extensive repository of tissue material with a long-term clinical follow-up, providing a valuable resource for translational clinical research.”

FFPE control tissue creates standardization and quality control. These tissues provide a stable reference point for experimental procedures, ensuring that tests are performed consistently across different laboratories over time.

Perhaps paraffin’s most useful duty is as a biomedical stabilizer. Researchers now have the ability to take smaller biopsy samples and examine the samples in great, transparent detail with the help of paraffin. Formalin-fixed Parafin-Embeded (FFPE) is a fantastic biomedical tool that prevents decay and enables the thorough examination of the tissue. Surrounding the sample with melted wax, or liquid paraffin, protects it from decay. FFPE is a preservation method that maintains the tissue sample’s composition—allowing researchers to examine thin, fragile layers without fear of damage. Samples preserved using FFPE also allow for the long-term storage of tissue samples and precise, reproducible results in histopathological research.

The use of paraffin, particularly FFPE tissue samples, mitigates every bioresearcher’s fear of encountering a false positive caused by a decaying sample. By studying FFPE tissue samples, scientists have made significant progress in analyzing genetic mutations, identifying targets for treatment, and predicting biomarkers. FFPE allows physicians to develop personalized approaches to treatment, which have led to better outcomes for patients.

Once you’ve gathered all the necessary material, you’re ready to prepare tissue samples for microscope slides. Whether you’re new to it or not, you likely understand how large of an undertaking tissue sample preparation is. That’s why we’re here to give you a complete step-by-step guide to preparing tissue samples for microscopic study. Below are the 5 steps you can follow to prepare tissue samples for microscope slides: 

Fix the biospecimens

After obtaining the fresh tissue sample, it must be fixated, whether that’s through freezing or chemical fixation. It’s important to start the fixation process immediately after collecting the biospecimen, ensuring that it holds its original structures and molecular makeup. In turn, you’ll get the best results! Fixation is critical for the rest of the tissue sample preparation. Fixation preserves the chemical composition of the biospecimen, securing and hardening the sample to initiate easy sectioning. Below are the two primary types of fixation that researchers use for tissue sample preparation:

• Fixative Solution: Researchers immerse biospecimens in a chemical fixative solution for 6–24 hours shortly after collection. Neutral Buffered Formalin or Paraffin-formalin are popular, effective solutions scientists and laboratories use for chemical fixation. Fixative solutions must penetrate every part of the biospecimen, preserving the sample and preparing it for microscopic analysis. 
• Freezing: Scientists submerge the tissue samples in a tissue-freezing medium, which is then immersed in liquid nitrogen. Freezing biospecimens is an alternative to the fixative solution method and is preferred by researchers who need an immediate diagnosis.

Fixing human tissue samples, whether by freezing or using a chemical fixative, assists in preserving specimens and preventing degradation. Once you fixate the biospecimens, they’re ready to be processed. 

Process the tissue samples

Tissue processing can be performed with an automated machine or by hand, preparing the biospecimen for sectioning. Here are three general steps involved in tissue processing:

1. Dehydration: Before converting the biospecimen into a solid form appropriate for sectioning, it must undergo dehydration. Ethanol is a popular agent used for the dehydration process. Ethanol removes water from the tissue sample and hardens it for microscopic use. Researchers immerse the specimen in multiple ethanol solutions of increasing intensity, ensuring that the tissue is free of water and formalin. 
2. Clearing: After the submergence of ethanol and before the embedment of wax, the tissue sample needs an in-between “clearing” medium that is compatible with both paraffin wax and ethanol. Xylene is a popular solvent that rids tissue of ethanol and prepares it for infiltration of wax. 
3. Embedding: Using an embedding center, the biospecimen is placed into a mold of molten wax (typically paraffin wax), forming what is called a “block” ready for sectioning. The newly created block is then cooled and prepared for thin-section cutting. Embedding also preserves the cellular structure of a tissue sample, making it more compatible with succeeding tissue sample preparation steps.

Processing helps preserve the integrity of tissue samples, leading to more accurate analysis and improving scientific diagnostics. Processed biospecimens also absorb stains more effectively and can be stored long-term in a biobank if needed. In summary, proper processing sets you up for success during the rest of the tissue sample preparation procedure.

Cut the biospecimens into sections

Now, your biospecimen is ready to be cut into sections on a microtome. First, the wax is removed from the surface of a block, exposing the tissue sample. Using a microtome, the biospecimen is sliced into sections no more than 4–5 micrometers. A microtome can cut continuously, creating a “ribbon” of tissue sections that are perfect for microscopy. If the sample is frozen, you’ll use a cryostat to cut it into tissue ribbons. These specimen ribbons can then be placed in a warm water bath to flatten. From here, tissue samples are easily collected for staining and examination on a microscopic slide. 

Stain the samples

Staining specimens is a vital element of tissue sample preparation. While there are numerous staining techniques, we’ll highlight a few of the most common methods below: 

• Hematoxylin and Eosin: The use of Hematoxylin and Eosin (H&E) is the most widely used staining technique for pathologists. Hematoxylin is a dye that stains acidic structures, while eosin is a counterstain done after hematoxylin that marks the sample’s basic structures. The result? Cell nuclei are often colored blue/purple, and other cellular structures that attract eosin (eosinophilic structures) are stained with a pink/red hue. RNA in ribosomes or the rough endoplasmic reticulum, for instance, would be stained blue, while the cytoplasm would be colored pink. 
• Gram Staining: Gram staining is used primarily to differentiate bacterial species according to the physical and chemical components of their cell walls. With the help of a gram stain, differing bacteria will change one of two different color sets (purple to blue or pink to red). Bacteria are then labeled as “gram-positive” or “gram-negative” according to their coloring. Gram-positive bacteria contain a hefty layer of peptidoglycan, making them appear purple. Gram-negative bacteria, on the other hand, hold a thin layer of peptidoglycan and other lipids in the cell wall, which washes out the violet in the decolorization process and stains them pink. Gram staining aids in diagnosing bacterial infections and the types of bacteria causing the illness.
• Masson’s Trichrome: Masson’s trichrome staining is a popular method that produces multicolor results on biospecimens. In a trichrome staining procedure, collagen is stained blue, muscle tissue is colored red, cytoplasm is dyed pink, and nuclei have a dark brown tint. The trichrome technique can distinguish collagen from muscle and identify pulmonary fibrosis, cardiac fibrosis, chronic kidney disease, muscular dystrophy, and various tumors of muscle origin.

Staining tissue samples enhances scientific studies by identifying a tissue sample’s components such as proteins, lipids, and carbohydrates. Varied stains can also aid in diagnosing diseases, differentiating normal and abnormal specimens, and detecting the presence and location of particular proteins. Overall, staining helps histologists better understand the minuscule structure and function of normal, malignant, and disease-state tissues. 

Mount the tissue sample sections for microscopic examination

After the biospecimen has been stained, the tissue sample section is ready to be mounted between a slide and coverslip, ensuring that it’s secure and ready for microscopic examination. Outlined below are the steps of mounting a slide: 

1. Apply a single drop of an aqueous-based or resinous mounting medium onto the tissue section.
2. Hold the coverslip at a 45° angle and allow the drop to spread to the edge of the slip. 
3. Gently let go of the slip so it covers the tissue section, allowing the medium to spread slowly and cover the biospecimen completely.

It’s important to utilize a clearing agent that’s compatible with the mounting medium, preventing issues in the mounting stage. It’s also necessary to label each slide with the patient ID, tissue type, and date. After mounting your biospecimen, it’s ready to be stored for observation under a microscope! Store tissue samples in a cool, dark place to prevent the fading of stains.

There is a diverse range of preservation methods within biospecimen storage. From freezing tissue samples in extremely low temperatures to fixating specimens in chemicals, tissue preservation is a learned science all on its own. However, the scientific community has relied on several popular preservation methods over the past few decades. Below are the 3 common preservation techniques scientists use for biospecimen storage: 

Snap-Frozen and Cryopreservation

Perhaps the most well-known preservation method is the act of freezing tissue samples. Scientists take one of two paths when freezing biospecimens: snap-freezing or cryopreserving. Let’s take a closer look at each of these preservation processes: 

Snap-freezing is the process of rapidly cooling tissue samples to temperatures below -70℃ using dry ice or liquid nitrogen. If using liquid nitrogen, researchers will place collected biospecimen samples on a cryomold or wrap them in foil, immerse them in the nitrogen, and immediately freeze them. On the other hand, researchers can use the dry ice method, placing the specimen on prepared dry ice. This method takes longer than liquid nitrogen and isn’t the preferred method. Snap-frozen tissue samples are stored in -80℃ freezers.

Cryopreservation is a long-term method of preserving samples at very low temperatures (-196℃ to -250℃) to preserve the overall integrity of cells and tissue. Cryopreservation involves the use of cryoprotectants (e.g., ethylene glycol, dimethyl sulfoxide, glycerol, etc.) to protect biospecimens from damage during the preservation process, specifically from ice crystals forming. 

Freezing tissue samples, whether through cryopreservation or snap-freezing, is a reliable preservation method used for long-term biospecimen storage. Tissue samples can be frozen for several years, maintaining their original structure and quality for an extended period.

Formalin-Fixed Paraffin-Embedded

Chemical fixation and embedding is another trusted preservation method for tissue samples. Formalin-fixed paraffin-embedded (FFPE) biospecimens are immersed in a fixative of 10% formalin. Formalin immediately stops the biological and cellular activity within specimens, stopping the decay process right after fixative immersion. After 18–24 hours, fixed specimens are then dehydrated using ethanol and prepared for embedment. The tissue samples are then embedded in paraffin wax, making it easier for the specimens to be sliced and mounted on microscopic slides. 

FFPE tissue samples can be stored indefinitely in biobanks, hospitals, laboratories, or research centers. For quality assurance reasons, fixed and embedded specimens should be stored at 4℃ with limited light exposure. FFPE tissues are compatible with a variety of histological staining techniques, such as immunohistochemistry (IHC), allowing researchers to find and locate protein structures within the biospecimen. Fixing and embedding tissue samples preserves the cellular and tissue structure of specimens, which is essential for histopathological studies. If stored at room temperature, FFPE samples can be stored for an extended amount of time, making it possible to curate extensive archives of specimens that can be used for future analysis. The fixing and embedding process of FFPE specimens is also well-standardized across the board, leading to consistently pure samples, which is paramount for comparative research.

Other Short-Term Techniques

Apart from freezing and FFPE techniques, there are other shorter-term preservation methods for biospecimen storage. Hypothermic preservation, for example, slows down the chemical processes occurring in human tissues and protects refrigerated specimens from cold-induced injuries. Tissue samples are bathed in a cold storage solution, preserving them for no longer than 1 to 2 days in low temperatures. 

Depending on the type of tissue sample, researchers can also store specimens in a refrigerator with a temperature of 2–8℃. Biospecimens stored in a refrigerator will stay preserved for just a few days, so it’s important to utilize these samples quickly for research purposes.

FFPE samples are a staple for modern research. The mainstream use of FFPE human tissue is for a scientific application called immunohistochemistry (IHC). IHC is one of the most common types of immunostaining. During the immunostaining process, tissue sections are mounted on a slide and soaked in a solution containing antibodies that bind to particular structures or proteins. IHC stains aid in visualizing antibodies which, in turn, reveal what antigens are present in the tissue sample. 

Information collected during IHC is commonly used to diagnose cancer, predict treatment response, and diagnose other medical conditions. For example, a healthcare provider may remove part of a tumor and send it to a commercial laboratory to test for cancer cells. Once received, a pathologist may use IHC techniques to study the tissue sample and determine the outcome. Other applications of FFPE human tissue include the following:

• Hematology: Researchers often use FFPE tissue samples to study blood-related disorders. Hematology is a vital field of research that’s led to the discovery of numerous cures for diseases caused by abnormalities in the blood. The study of hematology also applies to tissue regeneration, genetics, and toxicology.
• Immunology: This area of analysis concerns the response of the immune system in both normal and diseased states. Examining FFPE specimens from an individual with an autoimmune disease can help researchers recognize how and why the disease started. Immunology also contributes to the development of treatment or therapy for those with an autoimmune disease. 
• Comparative: Comparative research involves FFPE tissue samples collected from 100% healthy donors to differentiate varying types of biospecimens. In addition to using disease tissue for analysis, healthy samples can also be used for a variety of research and development purposes. This approach expands research beyond primarily the study of disease and enables a larger understanding of human biology.

FFPE tissue samples have become a favorite for researchers and laboratories worldwide. However, this isn’t the only preservation technique out there. Fresh frozen specimens are also trusted by scientific researchers around the globe. Let’s take a look at the main differences between these protective measures.

Without biobanks, researchers wouldn’t be able to perform transformative studies that improve human health. Biobanking allows the scientific industry to answer challenging research questions. Not only that, but biorepositories expand our understanding of the human body when it comes to health and disease; stored samples contribute to discovering new and improved ways to treat a vast variety of medical conditions. It’s a well-known fact that health research can take years to complete and an expansive number of people donate their samples and data to get impactful results. However, what if we told you that biobanks can drastically cut down the time and effort it takes to perform research? Biorepositories immediately speed up scientific studies by having samples and additional data available upon researchers’ requests. 

For decades, biospecimen research has resulted in new tests for diagnosing diseases, the development of more effective treatments, and increased quality of life for individuals experiencing chronic illness. There is still so much to be discovered within the health industry, and biobanks play a foundational part in contributing to the discovery of life-altering answers and the improvement of healthcare overall for years to come.