Untitled Quiz

Immunohistochemistry (IHC)

• IHC is a lab technique used in histology and pathology to visualize specific antigens in tissue sections.
• It relies on antigen-antibody interactions.
• Crucial for diagnosing diseases, especially cancers.
• Helps understand tissue biology.

Objectives

• Understand the fundamental principles of IHC.
• Learn the steps in the IHC process.
• Explore clinical and research applications of IHC.
• Discuss the interpretation of IHC results.
• Basic principles of immunohistochemistry

Antigens and Antibodies

• Antigens: Molecules (often proteins or glycoproteins) recognized by the immune system, present in tissues.
• Antibodies: Proteins produced by B cells; specifically bind to antigens.
  • Monoclonal or polyclonal in IHC.

Mechanism of IHC

• Binding: Primary antibody binds to the target antigen in the tissue.
• Detection: Secondary antibody (linked to an enzyme or fluorophore) binds to the primary antibody.
• Visualization: Detection system produces a measurable signal, enabling microscopic visualization.

Tissue Preparation

• Fixation: Preserves tissue morphology & antigenicity (commonly using formalin).
• Embedding: Tissues embedded in paraffin for thin sectioning.
• Sectioning: Thin slices (typically 4-5 μm) are cut and placed on slides.
• Deparaffinization and Rehydration: Removing paraffin and rehydrating sections with xylene and graded alcohols.

Antigen Retrieval

• Heat-induced Epitope Retrieval (HIER) or enzymatic retrieval unmasks antigens masked during fixation.

Blocking

• Blocks endogenous enzyme activity (e.g., peroxidase) and non-specific binding sites using serum or specific blocking agents.

Primary Antibody Incubation

• Tissue sections incubated with the primary antibody specific to the target antigen.

Secondary Antibody Incubation

• After washing, the sections are treated with a secondary antibody that recognizes the primary antibody.

Detection

• Adding a substrate that reacts with the enzyme linked to the secondary antibody produces a colorimetric or fluorescent signal.

Counterstaining

• Tissues often counterstained with Hematoxylin for contrast and to visualize tissue architecture.

Microscopy

• Slides examined under a microscope, and the staining pattern is analyzed.

Applications of Immunohistochemistry

• Cancer Diagnosis: Identifying tumor types, grades, and markers (e.g., hormone receptors).
• Research: Studying protein expression, localization, and cellular interactions in various biological contexts.
• Autoimmune Diseases: Identifying autoantibodies and understanding disease mechanisms.
• Infectious Diseases: Detecting pathogens in tissue sections for diagnosis (e.g., viral infections).

Interpretation of IHC Results

• Staining Patterns: Presence, intensity, and localization of staining provide diagnostic information.
• Positive and Negative Controls: Essential for validating staining results.
• Quantification: Some studies quantify staining intensity, correlating it with clinical outcomes.

Limitations and Challenges

• Specificity: Cross-reactivity can lead to false positives.
• Standardization: Protocol variability may affect reproducibility.
• Tissue Quality: Poorly prepared samples yield unreliable results.

Conclusion

• IHC is a vital tool, providing crucial insights for diagnosing and understanding various diseases.
• Technology advancements continuously enhance its clinical and laboratory applications.