Immunohistochemistry-in-Animal-Histology.pptx

PPT imunohistokimia

1. Immunohistochemistry in Histology Najwa Namira

2. What is Immunohistochemistry (IHC)? Definition & Principles IHC uses antibody-antigen specificity to localize target antigens (proteins) within tissue sections, visualized via light microscopy. Bridging Disciplines Combines immunology (antibody-antigen binding), histology (tissue structure), and chemistry (detection reactions). Preserving Context Unique in preserving native tissue microstructure, showing exact protein spatial distribution for comprehensive understanding. IHC identifies specific proteins by applying antibodies to prepared histological sections, making the antigen-antibody complex visible.

3. Methodologies: Tissue Preparation Fixation Methods Perfusion Fixation: Ideal for organs like the brain, removes blood, yields superior morphology. Immersion Fixation: For dissected tissues (<3mm thick), using PFA or NBF, typically overnight at 4°C. Sectioning Formats FFPE Tissue: Most common for long-term preservation, stable at room temperature. Frozen Tissue Sections: Preferred for sensitive antigens, snap-frozen and stored at -70°C. Meticulous tissue preparation and fixation are foundational for successful IHC, preserving morphology and antigenicity.

4. Methodologies: Antigen Retrieval & Antibodies Antigen Retrieval (AR) Crucial pre-treatment to unmask epitopes hidden by formalin fixation. HIER: Heat-Induced Epitope Retrieval (most common), breaks protein cross-links. PIER: Enzyme Digestion, uses proteolytic enzymes, risk of over-digestion. Antibody Selection High-quality antibodies are essential for specific detection. Primary Antibodies: Bind directly to target antigen (monoclonal or polyclonal). Secondary Antibodies: Visualize primary antibody complex, conjugated to a detectable marker. Optimization & Controls Careful titration, incubation, and blocking are vital. Positive and negative controls ensure accuracy.

5. Detection & Visualization Systems The choice of detection method impacts sensitivity and signal amplification. Indirect methods are preferred for their superior sensitivity. Making Antigens Visible Direct Method: Primary antibody is directly labeled. Quick, but less sensitive Indirect Method: (Most Common) Uses a labeled secondary antibody that binds to the primary antibody. Offers high sensitivity and signal boost. Common Detection Types Chromogenic Detection : Uses enzymes (HRP/AP) to create a colored, visible product. Examples: DAB (brown/black, permanent), AEC (red, needs aqueous mounting). Fluorescent Detection (IF): Uses fluorescent dyes (fluorochromes) that light up under a fluorescence microscope. Offers high sensitivity and allows for seeing multiple targets at once (multiplexing).Limitation: Fluorochromes can fade over time (photobleaching). Signal Amplification: Advanced systems (e.g., Polymer-based, TSA) further boost the signal for low-abundance antigens. Uses gold particles enhanced with silver for a visible metallic signal.

6. Counterstaining, Mounting, & Interpretation The choice of detection method impacts sensitivity and signal amplification. Indirect methods are preferred for their superior sensitivity. Counterstaining: Applied after IHC to provide visual contrast and highlight general cell structures (e.g., nuclei, cytoplasm). Hematoxylin: Stains nuclei dark blue/purple, providing excellent contrast. Eosin: Stains cytoplasm and extracellular matrix pink/red. DAPI: A fluorescent stain for nuclei, used in immunofluorescence . Mounting: Protects stained tissue and enables long-term storage and clear microscopic examination. Choice of mounting media (organic/aqueous) is crucial for chromogenic stains. Anti-fade media are vital for fluorescent stains. Special slides ensure tissue remains attached. Microscopic Interpretation Cellular Localization: Where the antigen is expressed within the cell. Tissue Distribution: Staining pattern across different cell types and structures. Labeling Intensity: Strength of the signal, indicating antigen abundance. Percentage of Positive Cells: Proportion of cells showing staining.

7. Applications in Pathology Oncology Aids in tumor classification, origin determination, and immunophenotyping for prognosis and treatment. Infectious Diseases Highlights and localizes viruses, bacteria, fungi, and protozoa not visible with routine staining. Neuroscience Maps distribution of neurotransmitters, receptors, and signaling molecules in animal brains. Toxicology & Drug Dev. Assesses drug efficacy/toxicity, studies metabolism, and evaluates gene delivery.

8. Advantages & Limitations Key Advantages Specificity & Localization: Identifies antigens in situ, preserving tissue microstructure. Diagnostic Utility: Enhances diagnoses, classification, and prognostication. Sample Versatility: Adaptable to fresh, frozen, or FFPE tissues. Current Limitations Antibody Availability: Lack of species-specific antibodies for all animals. Interpretation Complexity: Requires strong understanding of tissue morphology and pathology. Standardization: Not universally standardized, affecting reproducibility.

9. Future Directions Multiplex IHC Simultaneous study of multiple proteins on a single slide, crucial for complex biological interactions. Quantitative Analysis Digital image analysis and AI for objective, precise quantification, moving beyond subjective assessment. Automation & Digital Pathology Increased throughput, consistency, and reduced human error, integrating with advanced computational analysis. IHC continues to evolve, driven by demand for precise diagnostics and deeper insights into complex biological systems.

10. Thank You!