Tissue Embedding in Histology: Techniques & Common Errors скачать в хорошем качестве

Tissue Embedding in Histology: Techniques & Common Errors

Tissue embedding is a critical step in histology that involves encapsulating biological specimens in a medium (usually paraffin) to support thin sectioning for microscopic examination. The quality of embedding directly impacts the integrity of tissue sections and, consequently, the accuracy of histological analysis. Here’s a guide to mastering tissue embedding techniques and avoiding common errors. Techniques for Tissue Embedding** 1. Fixation: Purpose: Preserves tissue structure by preventing decomposition. Method: Typically done using formalin (10% neutral buffered formalin is common). Ensure tissue is adequately fixed before proceeding to dehydration. Best Practices: Avoid incomplete fixation, especially for larger or denser tissues. Fix tissue for at least 24-48 hours for optimal results. 2. Dehydration: Purpose: Removes water from tissue, which is incompatible with embedding media like paraffin. Method: Use graded alcohols (70%, 80%, 95%, and 100% ethanol) to dehydrate tissue incrementally. Best Practices: Ensure adequate time in each alcohol concentration to avoid incomplete dehydration, which can result in soft, poorly infiltrated tissue. 3. Clearing: Purpose: Replaces alcohol with a clearing agent (e.g., xylene) that is miscible with both ethanol and paraffin. Method: Immerse tissue in xylene (or alternatives like toluene). Best Practices: Ensure proper clearing time; if incomplete, tissues may appear opaque and poorly infiltrated. 4. Paraffin Infiltration: Purpose: Ensures tissue is completely permeated with paraffin wax, providing structural support for sectioning. Method: Typically performed at 60°C in an embedding center. Tissue is immersed in molten paraffin for 1-2 hours. Best Practices: Avoid overheating, as prolonged exposure to high temperatures can cause tissue shrinkage and hardening. 5. Embedding in Paraffin: Purpose: Embedding tissues in a paraffin block for microtome sectioning. Method: Once infiltrated with paraffin, tissues are positioned in a mold and allowed to solidify in a cold plate (usually at -5°C to 0°C). Best Practices: Orient tissues correctly for optimal sectioning (e.g., place epithelial tissues with the surface to be sectioned facing downward). 6. Sectioning: Purpose: Cut thin tissue sections (3-5 microns) for microscopic analysis. Method: Use a microtome to slice thin sections. Best Practices: Maintain a sharp blade and proper angle to avoid uneven sections. Collect ribbons of tissue without tears or folds. --- Common Errors and How to Avoid Them 1. Incomplete Fixation: Error: Results in autolysis (tissue degradation) or poor tissue preservation. Solution: Ensure tissue samples are thin enough for fixative to penetrate, and allow adequate fixation time. 2. Inadequate Dehydration: Error: Leads to poorly infiltrated tissues that crumble or cannot be sectioned. Solution: Use graded alcohols and ensure the tissue spends enough time in each concentration to remove all water. 3. Incomplete Clearing: Error: Tissues remain opaque or shrink during infiltration. Solution: Use sufficient clearing time, and ensure the clearing agent is fresh and compatible with your embedding medium. 4. Overheating During Infiltration: Error: Overheating can cause tissue hardening, shrinkage, and distortion. Solution: Maintain paraffin temperature between 55-60°C and avoid prolonged exposure to heat. 5. Incorrect Tissue Orientation: Error: Improper orientation results in sections where critical areas of the tissue are missed. Solution: Carefully orient the tissue in the mold, ensuring the area of interest is sectioned first. 6. Air Bubbles in Embedding Medium: Error: Air bubbles interfere with tissue structure and may prevent proper sectioning. Solution: Gently press down on the tissue during embedding to eliminate air bubbles and ensure complete contact with paraffin. 7. Paraffin Block Too Soft or Too Hard: Error: If the paraffin is too soft, sections may wrinkle; if too hard, sections may crack. Solution: Adjust paraffin mixture or use a different grade of paraffin suitable for your tissue type and environment. 8. Folds or Wrinkles in Sections: Error: Tissues wrinkle during sectioning or mounting, affecting microscopic evaluation. Solution: Stretch the tissue ribbon on a warm water bath and use gentle handling to prevent folds. Conclusions Mastering tissue embedding requires meticulous attention to each step of the process. By ensuring proper fixation, dehydration, clearing, and paraffin infiltration, and by avoiding common errors such as improper orientation or overheating, you can produce high-quality tissue sections for reliable histological analysis.