Tissue preparation for histology is a multi-step process that transforms delicate biological samples into durable, thin sections suitable for microscopic examination. The primary goal is to preserve tissue structure, preventing degradation and allowing for clear visualization of cellular details.
Steps in Tissue Preparation for Histology
The typical process involves the following key stages:
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Fixation:
- Purpose: To preserve tissue structure and prevent autolysis (self-digestion) and putrefaction (decay by bacteria).
- Method: The most common fixative is formalin (a solution of formaldehyde). Formalin cross-links proteins, stabilizing the tissue architecture. Other fixatives include glutaraldehyde, alcohol-based fixatives, and picric acid-based solutions. The choice of fixative depends on the specific tissue type and the intended staining techniques. Immersion fixation (submerging the tissue in fixative) or perfusion fixation (injecting fixative into the blood vessels) are common methods.
- Example: Organs removed during surgery are immediately placed in formalin to prevent degradation.
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Trimming and Cassette Loading:
- Purpose: To obtain representative tissue samples and prepare them for subsequent processing.
- Method: Fixed specimens are carefully trimmed to a manageable size and placed into labeled cassettes. These cassettes are small, perforated plastic containers that protect the tissue during processing.
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Dehydration:
- Purpose: To remove water from the tissue, as water is incompatible with paraffin wax, the embedding medium.
- Method: The tissue is passed through a series of increasing concentrations of alcohol (e.g., 70%, 90%, and 100% ethanol). Each step gradually replaces the water in the tissue with alcohol.
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Clearing:
- Purpose: To remove the alcohol from the tissue and replace it with a solvent that is miscible with both alcohol and paraffin wax. This makes the tissue translucent.
- Method: Common clearing agents include xylene, toluene, and limonene. These solvents dissolve the alcohol and prepare the tissue for infiltration with paraffin wax.
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Infiltration/Embedding:
- Purpose: To provide support and rigidity to the tissue, allowing for thin sectioning.
- Method: The tissue is infiltrated with molten paraffin wax under vacuum. The wax replaces the clearing agent and permeates the tissue. The tissue is then embedded in a block of paraffin wax. This process involves placing the tissue-filled cassette into a mold, filling the mold with molten paraffin, and allowing it to cool and solidify.
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Sectioning (Microtomy):
- Purpose: To cut extremely thin slices (sections) of the embedded tissue.
- Method: A microtome, a specialized instrument, is used to cut sections that are typically 3-10 micrometers thick. The paraffin block is mounted on the microtome, and a sharp blade is used to shave off thin sections.
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Mounting:
- Purpose: To adhere the thin tissue sections to a glass slide for viewing under a microscope.
- Method: The tissue sections are carefully floated on a warm water bath to flatten them, then picked up onto a glass slide. The slides are then dried.
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Staining:
- Purpose: To enhance contrast and differentiate cellular structures.
- Method: The most common staining method is hematoxylin and eosin (H&E) staining. Hematoxylin stains acidic structures (e.g., nuclei) blue/purple, while eosin stains basic structures (e.g., cytoplasm) pink/red. Other staining techniques, such as immunohistochemistry (IHC) and special stains (e.g., Masson's trichrome, Periodic acid-Schiff (PAS)), are used to highlight specific molecules or structures. Before staining, paraffin wax is typically removed by using xylene or other solvents, and the tissue is rehydrated by passing it through a series of decreasing concentrations of alcohol.
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Coverslipping:
- Purpose: To protect the stained tissue section and improve image quality.
- Method: A coverslip is mounted over the stained tissue section using a clear adhesive medium.
By following these steps, tissue samples are meticulously prepared to reveal their intricate cellular and structural details under microscopic examination, enabling accurate diagnosis and research.
