Understanding where and how proteins are expressed within tissues is fundamental to modern biology and pathology. Immunohistochemistry (IHC) provides a powerful bridge between molecular and cellular analysis by using antibody-based detection to localize specific proteins directly in tissue sections. Since its development in the 1940s, IHC has become indispensable in research and clinical diagnostics—enabling cancer classification, biomarker discovery, and drug target validation.

Principle of Immunohistochemistry

IHC is based on the specific binding between an antibody and its antigen. In this method, a primary antibody recognizes the target protein within fixed tissue. A secondary antibody, linked to a detectable label such as an enzyme or fluorophore, then binds to the primary antibody. The resulting signal can be visualized under a microscope, revealing the protein’s localization and abundance.

Workflow of Immunohistochemistry

1. Tissue Fixation and Embedding

   • Tissues are preserved using formalin to stabilize proteins and prevent degradation.

   • Samples are then embedded in paraffin wax and sectioned into thin slices (3–5 µm).

2. Deparaffinization and Rehydration

   • Paraffin is removed with xylene or substitutes, followed by graded ethanol washes to rehydrate the tissue.

3. Antigen Retrieval

   • Formalin fixation can mask antigenic sites.

   • Heat-Induced Epitope Retrieval (HIER) or Enzymatic Retrieval restores epitope accessibility for antibody binding.

4. Blocking Non-Specific Binding

   • Non-specific interactions are reduced by incubating tissue with a blocking buffer (e.g., serum or BSA).

5. Primary Antibody Incubation

   • The primary antibody binds specifically to the target protein.

   • Selection of an appropriate antibody (monoclonal vs. polyclonal) and optimal dilution is critical.

6. Secondary Antibody and Detection

   • The secondary antibody recognizes the primary antibody’s species and is conjugated to an enzyme (e.g., horseradish peroxidase, HRP) or a fluorescent dye.

   • Enzyme-based systems convert chromogenic substrates (e.g., DAB → brown precipitate) into visible signals.

7. Counterstaining and Mounting

   • Nuclei are counterstained (e.g., with hematoxylin) for contrast, and slides are mounted for microscopic evaluation.

Visualization and Detection Systems

Antibody Selection and Validation

Choosing a high-quality antibody is essential for specificity and reproducibility.

• Monoclonal antibodies: Highly specific; recognize a single epitope.

• Polyclonal antibodies: Detect multiple epitopes; higher sensitivity but may increase background.

• Validation: Includes western blotting or peptide blocking controls to confirm antigen specificity.

Applications of Immunohistochemistry

1. Cancer Diagnostics

   • Identifies tumor origin and subtype (e.g., ER, PR, HER2 in breast cancer).

   • Distinguishes between benign and malignant lesions.

2. Neuroscience and Developmental Biology

   • Visualizes neuronal markers and developmental proteins.

3. Infectious Disease Pathology

   • Detects pathogens (viruses, bacteria, fungi) within tissue sections.

4. Biomarker Discovery and Drug Research

   • Evaluates protein targets and downstream signaling in preclinical studies.

Optimization and Troubleshooting

Clinical and Research Relevance

In clinical diagnostics, IHC is essential for personalized medicine. For instance, assessing PD-L1 expression helps guide immunotherapy decisions, while Ki-67 indicates proliferative activity in tumors. In research, IHC provides spatial context to molecular data, linking gene expression with tissue morphology—something RNA-based assays alone cannot offer.

Conclusion

Immunohistochemistry remains one of the most insightful techniques for studying protein expression within the native tissue architecture. By combining molecular specificity with spatial resolution, IHC bridges the gap between genomics and histopathology. Continuous improvements in antibody design, multiplexing, and imaging technologies promise to further enhance the precision and power of IHC in both research and clinical applications.