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Hindi Editorial, Int J Ophthalmic Pathol Vol: -13 Issue: -1
Immunohistochemistry (IHC): Principles, Techniques and Applications
Pooja Yadav*
Department of Optometry, AIIMS, New Delhi, India
- *Corresponding Author:
- Pooja Yadav
Department of Optometry, AIIMS, New Delhi, India
E-mail: yadav212@gmail.com
Received: 01-Feb-2025, Manuscript No. iopj-25-169433; Editor assigned: 4-Feb-2025, Pre-QC No. iopj-25-169433 (PQ); Reviewed: 19-Feb-2025, iopj-25-169433; Revised: 26-Feb-2025, Manuscript No. iopj-25-169433 (R); Published: 30-Feb-2025, DOI: 10.4172/2324-8599.1000047
Citation: Pooja Y (2025) Immunohistochemistry (IHC): Principles, Techniques and Applications. Int J Ophthalmic Pathol 13:047
Introduction
Immunohistochemistry (IHC) is a powerful laboratory technique used to detect specific antigens (proteins) in tissue sections by exploiting the principle of antigen-antibody binding. It combines histological, immunological, and biochemical methods, making it an indispensable tool in pathology and research. IHC helps visualize the distribution and localization of biomarkers, enabling disease diagnosis, prognosis, and therapeutic decision-making, particularly in oncology.
Immunohistochemistry (IHC) is a widely used technique in pathology and biomedical research that enables the detection and localization of specific antigens (proteins) within tissue sections using antigen-antibody interactions. Combining principles of immunology, histology, and biochemistry, IHC plays a critical role in the diagnosis, classification, and prognosis of various diseases, particularly cancers. Since its introduction in the 1940s and the development of enzyme-labeled antibodies in the 1970s, IHC has become an indispensable tool in both clinical and experimental pathology [1].
The technique involves the application of specific antibodies to tissue sections, typically prepared from formalin-fixed, paraffin-embedded (FFPE) samples. These antibodies bind selectively to their target antigens, and the resulting antigen-antibody complexes are visualized using chromogenic or fluorescent detection systems. Common enzymes used in IHC include horseradish peroxidase (HRP) and alkaline phosphatase (AP), which produce a colored precipitate upon reaction with appropriate substrates. This allows for microscopic examination of the distribution and intensity of antigen expression within the context of preserved tissue architecture [2].
IHC offers several advantages, including high specificity, sensitivity, and the ability to study protein expression while maintaining cellular and tissue morphology. It is routinely employed to identify tumor origin, differentiate between benign and malignant lesions, detect infectious agents, and evaluate prognostic and predictive biomarkers such as HER2, Ki-67, and p53. Additionally, IHC contributes to understanding disease mechanisms and validating molecular targets in research settings [3].
Despite its strengths, IHC requires careful optimization and validation to avoid non-specific staining and ensure reproducibility. The quality of antibodies, tissue handling, and detection methods all significantly influence the outcome. With ongoing advancements in antibody technology, automation, and digital pathology, IHC continues to evolve as a powerful and reliable diagnostic and research tool in modern medicine [4].
Principle of Immunohistochemistry
The core principle of IHC is the specific binding of an antibody to its target antigen in biological tissues. This binding is then visualized using various detection systems, typically involving enzymes or fluorophores conjugated to the antibody. When exposed to a suitable substrate, enzyme-linked antibodies catalyze a color-producing reaction that can be seen under a microscope [5,6].
There are two main types of antibodies used in IHC:
Primary antibodies: These bind directly to the antigen of interest.
Secondary antibodies: These bind to the primary antibody and are usually conjugated to an enzyme or fluorophore for detection [7].
Steps in Immunohistochemistry
The IHC process involves multiple critical steps, each requiring optimization for reliable results:
Tissue Collection and Fixation
Tissues are typically fixed in 10% neutral-buffered formalin to preserve cellular morphology and antigenicity. Proper fixation is essential; under-fixation may result in poor preservation, while over-fixation can mask antigens [8].
Tissue Embedding and Sectioning
Fixed tissues are embedded in paraffin wax and sectioned into thin slices (usually 4??6 μm thick) using a microtome. The sections are then mounted on microscope slides.
Deparaffinization and Rehydration
Paraffin is removed using xylene or a substitute, followed by rehydration through a graded series of alcohol solutions to water [9].
Antigen Retrieval
Formalin fixation can mask antigenic sites. Antigen retrieval methods, such as heat-induced epitope retrieval (HIER) or enzymatic digestion, help unmask these sites for antibody access.
Blocking
To prevent non-specific binding, tissue sections are incubated with blocking solutions (usually containing serum or BSA) to block endogenous proteins or enzymes [10].
Antibody Incubation
The primary antibody is applied to the tissue and incubated for a specific period. This is followed by incubation with a labeled secondary antibody if the primary is not directly conjugated.
Detection and Visualization
The most common detection method uses an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), which reacts with a chromogenic substrate (e.g., DAB) to produce a colored precipitate at the antigen site.
Counterstaining and Mounting
A counterstain, typically hematoxylin, is applied to visualize cell nuclei. Finally, the slide is mounted with a coverslip for microscopic examination.
Types of Detection Systems
IHC detection systems can be broadly categorized into:
Chromogenic Detection: Produces a colored end-product visible under light microscopy. DAB (3,3?-diaminobenzidine) is a commonly used chromogen, producing a brown stain.
Fluorescent Detection: Uses fluorophore-conjugated antibodies and is observed under a fluorescence microscope. This method allows multiplex staining but requires specialized equipment.
Common IHC Markers
Different markers are used to identify specific cell types, disease states, or tissue origins. Some commonly used IHC markers include:
CD markers (e.g., CD3, CD20) for lymphocytes
Cytokeratins (e.g., CK7, CK20) for epithelial cells
Vimentin for mesenchymal origin
HER2/neu in breast cancer
p53 and Ki-67 for cell proliferation and tumor suppressor status
Applications of IHC
Cancer Diagnosis and Classification
IHC plays a vital role in distinguishing between tumor types, subtypes, and tissue of origin. For instance, distinguishing primary lung adenocarcinoma from metastatic lesions may depend on TTF-1 and Napsin A staining.
Prognostic and Predictive Markers
Certain IHC markers can predict disease outcome and guide treatment choices. For example, HER2 positivity in breast cancer suggests eligibility for trastuzumab therapy.
Infectious Disease Detection
IHC can detect pathogens like viruses, bacteria, and fungi within tissue sections, aiding in diagnosing infections such as cytomegalovirus or tuberculosis.
Neuropathology
IHC helps identify specific proteins like tau, β-amyloid, and alpha-synuclein in neurodegenerative diseases.
Research Applications
In developmental biology, neuroscience, and immunology, IHC is used to map protein expression and understand cellular behavior and interactions.
Advantages of IHC
High specificity and sensitivity due to antibody-antigen binding
Morphological context is retained, allowing localization within tissue
Versatility in detecting a wide range of molecules
Permanent records in chromogenic methods for archival purposes
Limitations and Challenges
Antibody specificity: Non-specific or poorly validated antibodies can lead to false results.
Antigen masking: Inadequate antigen retrieval can affect sensitivity.
Subjective interpretation: IHC results often require expert pathological evaluation.
Standardization: Variability in protocols between labs can affect reproducibility.
Recent Advances
Advancements in IHC include multiplex immunohistochemistry, which allows simultaneous detection of multiple targets using different fluorophores. Digital pathology and AI-based image analysis are improving the accuracy and consistency of IHC interpretation. Additionally, standardized protocols and automation are reducing variability and improving throughput in clinical settings.
Conclusion
Immunohistochemistry remains a cornerstone of modern pathology, bridging molecular biology and diagnostic histology. Its ability to localize specific proteins within complex tissue environments makes it invaluable for disease diagnosis, prognosis, and biomedical research. As technology evolves, IHC continues to expand its capabilities, offering deeper insights into the molecular underpinnings of health and disease.
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