EN
Fixation is a cornerstone of tissue processing, essential for preparing biological samples for microscopic examination in histology. This critical process preserves tissue structure, prevents degradation, and enables detailed analysis of cellular and molecular features, driving advancements in medical research and diagnostics. Whether you’re a lab technician, biomedical researcher, or pathologist, mastering fixation techniques and best practices is vital for achieving high-quality results. In this comprehensive guide, we explore what fixation entails, its purpose, the types of fixatives used, and key protocols and timing considerations. Read on to unlock the secrets of successful fixation in histology.
Fixation marks the first step in tissue processing, where a chemical or physical agent, known as a fixative, is applied to a fresh biological sample to preserve its structure and composition. Fresh tissues, collected from patients or experimental animals, are prone to autolysis—where endogenous enzymes break down cellular components—and putrefaction, where bacteria cause decomposition. Fixation halts these degradative processes by stabilizing proteins, lipids, and other cellular elements, maintaining a state as close as possible to in vivo conditions. This preservation is crucial for subsequent steps like dehydration, clearing, infiltration, and embedding, which prepare tissues for thin sectioning and microscopic analysis. By arresting decay and hardening tissues, fixation ensures researchers and clinicians can study cellular details, diagnose diseases, and advance biomedical science with precision and reliability.
Fixation serves multiple essential functions in histology, going beyond simple preservation to ensure quality outcomes. It stabilizes the three-dimensional structure of tissues by cross-linking proteins, preventing collapse or distortion during later processing stages, which is vital for observing cellular morphology under a microscope. This process also inhibits autolysis and bacterial growth, safeguarding tissues from enzymatic and microbial breakdown that could obscure critical features. Additionally, fixation enhances tissue hardness, making samples easier to handle and cut into thin, consistent sections via microtomy. Another key role is retaining chemical reactivity, allowing specific staining techniques, such as hematoxylin and eosin (H&E), to highlight cellular structures or molecules for analysis. Without effective fixation, tissues degrade, undermining the reliability of histological studies and diagnostic results, making this step indispensable in histology workflows.
A range of fixatives are employed in histology, each with distinct properties tailored to specific tissues, research goals, or diagnostic needs. The choice of fixative directly impacts preservation quality, staining compatibility, and downstream analysis. Below, we explore the most common types used in tissue processing.
Formalin, a 10% neutral buffered formaldehyde solution, is the gold standard in histology. It penetrates tissues effectively, cross-links proteins to preserve structure, and works well with a wide range of stains, making it ideal for routine medical and research applications. Its phosphate buffering prevents pH shifts that could harm tissues, ensuring consistent results for paraffin-embedded sections. However, prolonged exposure can over-harden tissues, so careful timing is essential.
Alcoholic fixatives, such as 70% ethanol or Carnoy’s solution (a blend of ethanol, chloroform, and acetic acid), are prized for rapid penetration and preservation of specific components like glycogen. Ethanol suits small, delicate samples, while Carnoy’s excels in fixing nucleic acids, popular in molecular studies. These fixatives also dehydrate tissues, but over-fixation can shrink or brittle samples, requiring close monitoring.
Glutaraldehyde, a potent cross-linking agent, is widely used in electron microscopy for its superior preservation of ultrastructure. It fixes proteins and lipids exceptionally well but penetrates slowly, making it less common for routine histology, as it can interfere with some stains and requires longer washing to remove excess. Other options include Bouin’s solution, combining picric acid, formalin, and acetic acid, which excels for soft tissues like embryos or testes but may degrade DNA. Mercuric chloride-based fixatives, like B-5, offer sharp nuclear detail for hematological samples but are toxic and less used due to safety concerns. Physical methods, such as freeze-drying or heat fixation, serve specialized cases, though chemical fixatives dominate histology. Choosing the right fixative depends on tissue type, study goals, and compatibility with subsequent steps.
Effective fixation demands well-designed protocols and precise timing to achieve optimal preservation without compromising tissue quality. Protocols vary based on tissue type, size, fixative choice, and intended analysis, while timing balances penetration and chemical reaction. Here are key considerations and best practices for successful fixation:
Immediate Fixation: Begin fixation immediately post-collection to prevent autolysis. Submerge tissues in a fixative like 10% neutral buffered formalin at a 10:1 fixative-to-tissue volume ratio to ensure thorough penetration, critical for preserving structure from the outset.
Tailored Timing: Small samples, such as biopsies less than 4mm thick, may fix adequately in 6-12 hours, while larger specimens, like organs, require 24 hours or more. Under-fixation leaves tissues soft and prone to degradation, while over-fixation with formalin or glutaraldehyde can over-harden, reducing staining affinity.
Temperature Control: Conduct fixation at room temperature (20-25°C) for most cases, as it’s effective and safe. Gentle heating, such as 37°C, can speed penetration for dense tissues, but avoid excessive heat to prevent damage or distortion to delicate structures.
Processor Integration: Automated tissue processing equipment often includes a fixation step as the first station, streamlining workflows for labs handling multiple samples. Use these to maintain consistency, especially for routine histology with formalin-fixed tissues.
Sample Preparation: Trim tissues to 4-5mm thickness for better fixative infiltration, especially for dense or fatty samples like brain or breast. Agitation or vacuum cycles in processors can enhance penetration, reducing fixation time without sacrificing quality.
Specialized Protocols: For electron microscopy, use 2.5% glutaraldehyde for 2-6 hours, followed by a buffer wash to preserve ultrastructure. For molecular studies, rapid fixation with Carnoy’s or ethanol (1-2 hours) protects DNA and RNA, suiting genetic analysis needs.
Post-Fixation Handling: After fixation, dissect tissues if needed to select areas for embedding, placing them in labeled cassettes for processing. This ensures accurate tracking and alignment for downstream sectioning and analysis.
Safety and Documentation: Adhere to safety guidelines, especially with toxic fixatives like mercuric chloride, to protect lab personnel. Document fixation times, fixative type, and conditions to monitor consistency and troubleshoot issues, ensuring reliable results.
HealthSky's liquid-based cytology equipment delivers advanced and efficient solutions for cervical cancer screening and cytological analysis. Using a specialized liquid medium, it preserves cellular samples for clearer, more consistent microscopic evaluation. The automated system ensures uniform sample preparation and faster turnaround times, making it a reliable choice for modern cytology laboratories focused on accuracy and productivity.
