What is Immunohistochemistry?
Immunohistochemistry (IHC) is a technique used in histology to detect specific antigens (proteins) within tissue sections by employing antibodies that bind to these antigens. The presence and localization of the antigen can then be visualized using various detection methods.
Preparation of tissue sections: Tissues are fixed, usually with
formalin, and embedded in paraffin. Thin sections are then cut and mounted onto slides.
Antigen retrieval: This step involves treating the tissue sections to unmask antigens that may have been altered during fixation. This can be done using heat or enzymatic methods.
Blocking: To prevent non-specific binding, sections are treated with a blocking solution.
Primary antibody incubation: The tissue sections are incubated with a primary antibody that specifically binds to the target antigen.
Secondary antibody incubation: A secondary antibody that binds to the primary antibody is then applied. This secondary antibody is usually conjugated to an enzyme or a fluorescent dye.
Detection: The enzyme or dye on the secondary antibody is used to produce a visible signal, which can be visualized under a microscope.
Enzymatic detection: The secondary antibody is conjugated to an enzyme such as
horseradish peroxidase (HRP) or
alkaline phosphatase (AP). A substrate for the enzyme is added, which produces a colored precipitate at the site of the antigen.
Fluorescent detection: The secondary antibody is conjugated to a fluorescent dye. When exposed to a specific wavelength of light, the dye emits fluorescence that can be detected using a fluorescence microscope.
Chromogenic detection: Similar to enzymatic detection, but uses chromogens that produce a visible color reaction.
Diagnostic pathology: IHC is used to identify specific cell types and subtypes in tissue samples, aiding in the diagnosis of diseases such as cancer.
Research: IHC is employed to study the distribution and localization of proteins within tissues, providing insights into cellular and molecular processes.
Prognostic markers: IHC can help determine the prognosis of certain diseases by identifying biomarkers associated with disease progression.
Specificity: Ensuring that antibodies specifically bind to their target antigen without cross-reacting with other proteins can be difficult.
Sensitivity: Detecting low-abundance proteins requires highly sensitive antibodies and detection methods.
Standardization: Variations in tissue preparation, antibody quality, and detection methods can affect the reproducibility and reliability of IHC results.
Multiplex IHC: Allows the simultaneous detection of multiple antigens in a single tissue section, providing more comprehensive data.
Digital pathology: The use of digital imaging and analysis tools to quantify IHC results, improving accuracy and reproducibility.
Automated IHC: Automation of IHC processes increases throughput and reduces variability between samples.
