Histology tissue preparation is a multi-step process that transforms raw biological samples into microscopic slides, enabling detailed examination of cellular structures and tissue architecture. This meticulous procedure ensures the preservation, stability, and visibility of tissues for accurate diagnosis and research.
The Journey from Tissue to Slide: Key Steps
Preparing tissue for histological analysis involves several critical stages, each designed to preserve the tissue, stabilize its components, and make it suitable for microscopic study. The primary goal is to create thin, transparent sections that can be stained and viewed under a microscope.
1. Fixation: Preserving the Tissue
The initial and most crucial step in histology tissue preparation is fixation. Immediately after collection, tissues must be immersed in a fixative solution to prevent degradation.
- Purpose:
- To stop all cellular processes, preventing autolysis (self-digestion) and putrefaction (bacterial decomposition).
- To harden the tissue, making it easier to handle and cut.
- To preserve the tissue's cellular and structural components as close to their living state as possible.
- To enhance the staining reaction of cellular elements.
- Common Fixative: The most widely used fixative is a formalin solution, typically 10% neutral buffered formalin. Formalin chemically cross-links proteins, effectively stabilizing the tissue's structure.
- Method: Tissues are placed in a container with a sufficient volume of fixative (usually 10-20 times the tissue volume) to allow for proper penetration.
2. Grossing and Cassetting: Organization and Trimming
Once adequately fixed, the specimens undergo grossing, a process performed by a pathologist or trained technologist.
- Grossing: This involves carefully examining, describing, and measuring the tissue.
- Trimming: Small, representative portions of the tissue, typically no larger than 20x25x4 mm, are precisely trimmed from the larger specimen. This ensures proper fixative penetration and allows for subsequent processing.
- Cassette Placement: The trimmed specimens are then placed into small, perforated plastic containers called labeled cassettes. Each cassette is uniquely identified, usually with a barcode or number, linking it directly to the patient or study. This step is vital for maintaining sample identity throughout the process.
3. Tissue Processing: Dehydration, Clearing, and Infiltration
After fixation and grossing, tissues are ready for automated tissue processing, which prepares them for embedding. This multi-stage process occurs in a tissue processor and involves:
- Dehydration: Water within the tissue must be completely removed, as paraffin wax (used later) is immiscible with water. This is achieved by passing the tissues through a series of ascending concentrations of alcohol (e.g., 70%, 90%, 100% ethanol).
- Clearing: Alcohol is also immiscible with paraffin wax, so it must be removed. Tissues are submerged in a "clearing agent," such as xylene. Xylene is miscible with both alcohol and paraffin, making the tissue appear translucent or "clear" as it replaces the alcohol.
- Paraffin Infiltration (Embedding Prep): Finally, the clearing agent is replaced by molten paraffin wax. Tissues are immersed in heated paraffin, which permeates all the spaces previously occupied by water and alcohol, providing structural support. This step is also sometimes referred to as infiltration.
4. Embedding: Creating a Solid Block
Embedding is the process of creating a solid block of tissue within paraffin wax.
- Process: The processed tissue, saturated with molten paraffin, is carefully oriented in a mold. Fresh, molten paraffin wax is then poured around it, and the entire mold is cooled, allowing the wax to solidify.
- Result: This forms a solid paraffin block, with the tissue firmly encapsulated within, providing the rigidity necessary for very thin sectioning. Proper orientation of the tissue in the block is critical to ensure that the relevant diagnostic features are visible in the final slide.
5. Sectioning: Slicing Thin Sections
The solidified paraffin block, containing the tissue, is then mounted onto a microtome.
- Microtome: This specialized instrument uses a sharp blade (often steel or glass) to cut extremely thin sections from the block.
- Thickness: Histology sections are typically cut at thicknesses ranging from 3 to 5 micrometers (µm), which is thinner than a human hair.
- Floating and Mounting: These ribbon-like sections are carefully floated onto a warm water bath to flatten them, and then picked up onto glass microscope slides. The slides are then dried to ensure the tissue adheres permanently.
6. Staining: Visualizing Structures
Once mounted on slides, the colorless tissue sections are ready for staining, which highlights cellular and extracellular components.
- Deparaffinization and Rehydration: Before staining, the paraffin wax must be removed (deparaffinization) using a solvent like xylene, and the tissue rehydrated by passing it through descending concentrations of alcohol back to water.
- Common Stains: The most common staining method is Hematoxylin and Eosin (H&E):
- Hematoxylin: Stains acidic components (like cell nuclei, ribosomes) a blue-purple color.
- Eosin: Stains basic components (like cytoplasm, collagen, muscle) pink or red.
- Special Stains and Immunohistochemistry: For specific diagnostic needs, various special stains (e.g., Masson's Trichrome for collagen, Periodic Acid-Schiff for carbohydrates) or immunohistochemical techniques (using antibodies to detect specific proteins) can be applied.
- Dehydration and Coverslipping: After staining, the slides are again dehydrated, cleared, and then a coverslip is applied with a mounting medium to protect the tissue and provide a clear viewing surface.
Overview of the Histology Workflow
The table below summarizes the main steps involved in preparing histology tissue for microscopic examination:
| Step | Purpose | Key Materials/Tools |
|---|---|---|
| Fixation | Preserve tissue, prevent degradation, harden tissue | Formalin solution |
| Grossing & Cassetting | Trim tissue, organize, maintain identity | Labeled cassettes |
| Dehydration | Remove water from tissue | Ascending alcohol series |
| Clearing | Remove alcohol, prepare for paraffin infiltration | Xylene or other clearing agent |
| Paraffin Infiltration | Infiltrate tissue with molten paraffin for support | Molten paraffin wax |
| Embedding | Form a solid block of tissue in paraffin | Embedding center, molds |
| Sectioning | Cut thin tissue slices (3-5 µm) | Microtome |
| Staining | Visualize cellular components | H&E stains, special stains |
| Coverslipping | Protect tissue, provide clear viewing | Mounting medium, coverslip |
This comprehensive process ensures that pathologists and researchers can accurately analyze tissue samples, leading to diagnoses, understanding disease progression, and advancing medical knowledge. For more detailed information, resources like Leica Biosystems and Bitesize Bio provide extensive guides on these techniques.
