Paraffin embedding is a method in histopathology used to preserve tissue samples for microscopic study. The procedure involves placing biological tissue into molten paraffin wax, which cools and hardens. This process creates a stable, solid block that can be thinly sliced for staining and analysis. The technique supports medical diagnostics and scientific research by enabling a detailed examination of cellular structures.
Understanding Tissue Preparation for Study
Fresh biological tissue is soft, fragile, and mostly water, making it difficult to slice into the thin sections needed for microscopic examination. Once removed from its environment, tissue is also highly susceptible to decomposition by enzymes and bacteria, a process known as autolysis and putrefaction. This decay rapidly degrades cellular integrity. To overcome these issues, tissue must be stabilized.
The primary goal is preservation, which halts decay and maintains the tissue’s original architecture. A secondary need is to give the soft tissue a firm consistency. This allows a specialized instrument called a microtome to cut sections that are typically only a few micrometers thick—much thinner than a human hair. This preparation is necessary to get a clear view of individual cells and their relationships.
The Step-by-Step Paraffin Embedding Process
The process begins with fixation and proceeds through several stages to prepare the tissue for sectioning.
Fixation
The first step is fixation, where the tissue is immersed in a chemical solution, most commonly formalin. Fixation preserves the tissue’s structure by cross-linking proteins and inactivating the enzymes that cause decay. This chemical process stabilizes the cellular components for subsequent steps.
Dehydration
Because paraffin wax is hydrophobic and will not mix with water, the tissue must be dehydrated. This is accomplished by passing the tissue through a series of baths with increasing concentrations of alcohol, such as ethanol. This graded process gently removes all water from the tissue without causing cellular distortion.
Clearing and Infiltration
Following dehydration, the alcohol is replaced with a clearing agent like xylene, which is miscible with both alcohol and paraffin. The now-cleared tissue is submerged in molten paraffin wax, kept at a temperature between 55 and 60 degrees Celsius. During this infiltration stage, the warm wax permeates the specimen, filling all spaces previously occupied by water.
Embedding
After infiltration, the tissue is ready for embedding, also called “blocking.” The specimen is carefully placed and oriented in a mold to ensure the correct plane is sectioned. Molten paraffin is poured into the mold to surround the tissue and then cooled on a cold plate, forming a solid block with the tissue inside that provides the necessary support for sectioning.
Visualizing Tissues After Embedding
After sectioning, the thin tissue slices are transparent and lack contrast, making cellular components difficult to see. To make these structures visible, the sections must be stained. First, a thin ribbon of tissue is mounted on a glass slide, the paraffin is dissolved with a solvent, and the tissue is rehydrated.
With the tissue rehydrated, stains can be applied. Staining uses a combination of dyes that selectively bind to different cellular structures based on their chemical properties. The most common staining method is Hematoxylin and Eosin (H&E). Hematoxylin is a basic dye that stains acidic structures, like the cell nucleus, a purplish-blue color, while Eosin is an acidic dye that stains basic structures, such as the cytoplasm, in shades of pink and red.
This differential staining creates the contrast needed to identify cell types, their morphology, and the overall tissue architecture. Once staining is complete, the section is dehydrated again, and a permanent mounting medium and a glass coverslip are applied. The coverslip protects the tissue and provides a flat surface for microscopic viewing.
Significance in Medical Diagnosis and Research
Paraffin embedding is important for medical diagnosis, particularly in pathology. When a patient has a biopsy or a tumor removed, the tissue is processed into paraffin blocks. Pathologists examine stained sections from these blocks to identify cellular abnormalities and diagnose diseases like cancer. Viewing the precise arrangement, size, and shape of cells allows for accurate tumor grading and staging, which guides treatment decisions.
The technique is also a tool in scientific and clinical research, allowing scientists to study the structure of normal tissues and organs in great detail. In disease research, it is used to understand how illnesses develop at a cellular level. For pharmaceutical development, researchers analyze tissues from preclinical studies to evaluate the safety and efficacy of new drugs, observing their effects on different organs. The archives of formalin-fixed, paraffin-embedded tissue blocks stored by hospitals are also a resource for retrospective studies, enabling investigation into diseases years after samples were collected.