Paraffin embedding is a fundamental technique used in histology and pathology laboratories. This process involves encasing biological tissue specimens within a solid, supportive matrix of paraffin wax. Its primary purpose is to provide rigidity and stability to delicate tissues, allowing them to be cut into extremely thin, consistent sections, typically 4 to 10 micrometers thick. These sections are then mounted on glass slides for microscopic examination, which is common for diagnosing diseases and conducting scientific research.
The Initial Step of Tissue Fixation
The initial stage in preparing tissue for microscopic study is fixation. Its main goal is to prevent self-digestion (autolysis) and bacterial decomposition (putrefaction), which begin immediately after tissue removal. This stabilizes the tissue’s structure, preserving its components. Formaldehyde-based fixatives are preferred for long-term tissue preservation and yield favorable results for various staining techniques.
The most common chemical fixative is 10% neutral buffered formalin (NBF), approximately 3.7% to 4.0% formaldehyde in a phosphate buffer at pH 7. This solution stabilizes proteins by forming cross-links, converting the tissue’s semi-fluid consistency into a semi-solid gel. Other fixatives, such as Bouin’s solution (containing picric acid, formaldehyde, and acetic acid), are used for specific tissue types like connective tissue or endocrine glands due to their ability to preserve certain cellular details. Alcohol-based fixatives, like ethanol, denature proteins by removing water and are used for specific molecular assays.
Several factors influence fixation quality, including tissue size, time before fixation, and fixative volume. Tissue thickness should be no more than 4-5 mm for proper fixative penetration. Tissues should be immersed in fixative immediately after collection, with the volume of fixative being at least 15-20 times the tissue volume. Adequate fixation time ranges from 6 to 24 hours for small biopsies, with larger samples often requiring overnight fixation for complete penetration and stabilization.
Tissue Processing for Wax Infiltration
After fixation, tissues undergo sequential processing steps to prepare them for infiltration with paraffin wax. This involves dehydration and clearing, which progressively remove water and introduce an intermediate solvent compatible with wax. Automated tissue processors are used for these steps, ensuring consistent and controlled processing.
Dehydration
Dehydration removes all water from fixed tissue, as paraffin wax is not miscible with water. This is achieved by immersing the tissue in a series of graded alcohol solutions, typically ethanol, of increasing concentrations. A common sequence involves baths of 70%, 95%, and then 100% ethanol, with multiple changes at higher concentrations for thorough water removal. Using a graded series gently removes water, preventing excessive cellular distortion or shrinkage from sudden exposure to high-concentration alcohol.
Clearing
Following dehydration, a clearing agent removes alcohol from the tissue. Since alcohol is not miscible with paraffin, an intermediate solvent is necessary. Common clearing agents include xylene or xylene substitutes like citrus-based solvents or isopropanol. These agents replace the alcohol within the tissue and impart optical clarity or translucency, hence the term “clearing.” The tissue is submerged in the clearing agent for multiple changes, ensuring complete displacement of alcohol and preparing the tissue for molten paraffin.
Paraffin Infiltration and Block Creation
With the tissue cleared, the next phase involves permeating it with molten paraffin wax, followed by the creation of a solid tissue block. This stage provides the necessary support for subsequent sectioning.
Infiltration
Infiltration involves submerging the cleared tissue in molten paraffin wax, within a tissue processor or heated bath. The wax, heated to temperatures between 56°C and 60°C, permeates the tissue, replacing the clearing agent and filling all interstitial spaces. This process allows the wax to fully impregnate the tissue, creating a homogeneous matrix. Careful temperature control is important during infiltration to avoid overheating the tissue, which can cause hardening or shrinkage, potentially damaging cellular components.
Embedding (Block Creation)
The final step in preparing the paraffin block is embedding. The infiltrated tissue is carefully removed from the molten wax and placed into a metal mold, which is then filled with fresh molten paraffin. Proper orientation of the tissue within the mold is important, as this determines the plane for microscopic sectioning. For instance, if a biopsy needs to show all tissue layers, it must be oriented on its edge. The mold, containing the tissue and molten wax, is then placed on a cold plate or cooling surface to allow the paraffin to rapidly solidify. Once cooled, the wax forms a solid block that supports the delicate tissue, making it ready for precise slicing on a microtome.
Addressing Common Embedding Problems
Even with careful adherence to protocols, issues can arise during paraffin embedding. Recognizing and addressing these problems helps ensure high-quality tissue sections for accurate analysis.
Brittle or “crumbly” tissue results from over-dehydration or excessive heat during wax infiltration. To mitigate this, ensure dehydration times are appropriate for tissue size and maintain paraffin temperatures between 56°C and 60°C. Spongy or soft tissue suggests incomplete dehydration or clearing, meaning water or alcohol remains. Adjusting the duration of alcohol and clearing agent baths can help achieve thorough removal.
“Chatter” or wrinkles in sections, appearing as wavy lines or folds, can be caused by improper processing or dull microtome blades during sectioning. Ensuring complete infiltration and using sharp blades can reduce this problem. Tissue shrinkage or distortion, where the tissue appears smaller or misshapen, may stem from improper fixation (e.g., inadequate fixative volume or duration) or overly aggressive processing steps like rapid dehydration. Following recommended fixative volumes and gradual dehydration sequences helps minimize these effects.