What is Sample Preparation in Histology?
Sample preparation in histology is a critical process that ensures tissues are properly preserved, sectioned, and stained for microscopic examination. It involves a series of steps designed to maintain the structural integrity and biochemical properties of the tissue.
Why is Fixation Important?
Fixation is the first and most crucial step in histology sample preparation. It involves treating the tissue with chemicals to preserve its structure and prevent degradation. Common fixatives include
formalin, glutaraldehyde, and alcohol-based solutions. Proper fixation ensures that cellular components remain intact and are not distorted during subsequent processing steps.
Dehydration: The tissue is gradually dehydrated using increasing concentrations of ethanol or other dehydrating agents.
Clearing: Dehydrated tissue is treated with a clearing agent like xylene, which makes the tissue transparent.
Infiltration: The cleared tissue is infiltrated with a medium such as paraffin wax, which supports its structure during sectioning.
How is Embedding Done?
Embedding involves placing the infiltrated tissue into a mold filled with embedding medium (usually paraffin wax or resin). The medium is then allowed to solidify, providing a support matrix for the tissue. This step is crucial for obtaining thin, uniform sections for microscopic examination.
What is the Purpose of Sectioning?
Sectioning is the process of cutting thin slices of embedded tissue using a microtome. These sections, typically 4-5 micrometers thick, are placed on glass slides. Thin sections are essential for detailed examination under a microscope, as they allow light to pass through and reveal fine cellular structures.
Why is Staining Necessary?
Staining enhances the contrast of tissue sections, making cellular components more visible under the microscope. Common stains include
hematoxylin and eosin (H&E), which stain nuclei blue and cytoplasm pink, respectively. Special stains and immunohistochemical techniques can also be used to highlight specific cellular components or markers.
How is Mounting Performed?
After staining, the sections are mounted on glass slides using a mounting medium and cover slip. This step preserves the sections and allows for long-term storage and examination. The mounting medium also improves the optical properties of the sample, enhancing visibility under the microscope.
What are Frozen Sections?
Frozen sectioning is an alternative to paraffin embedding, often used for rapid diagnosis during surgery. In this method, tissues are quickly frozen using a cryostat and sectioned while still frozen. Although faster, frozen sections may not provide the same level of detail as paraffin-embedded sections but are invaluable for intraoperative consultations.
What are Artefacts and How to Avoid Them?
Artefacts are distortions or anomalies introduced during sample preparation that can obscure or mimic pathological findings. Common artefacts include air bubbles, folds, and improper fixation. Adhering to meticulous techniques and using high-quality reagents can minimize artefact formation, ensuring accurate diagnosis.
