What is Immunohistochemistry?
Immunohistochemistry (IHC) is a powerful technique used in
histology to visualize the presence and location of specific
proteins in tissue sections. By utilizing antibodies that bind to target antigens, IHC allows researchers and clinicians to observe the distribution and abundance of these proteins within the tissue context.
Sample Preparation: Tissues are fixed, usually with formalin, and then embedded in paraffin to preserve structure and antigenicity.
Sectioning: Thin sections of the tissue are cut using a microtome and mounted onto slides.
Antigen Retrieval: This step, often involving heat or enzymatic treatment, is crucial to unmask antigens that may have been obscured during fixation.
Blocking: Non-specific binding sites are blocked to reduce background staining.
Primary Antibody Incubation: The tissue sections are incubated with a primary antibody specific to the target antigen.
Secondary Antibody Incubation: A secondary antibody, which binds to the primary antibody, is applied. This secondary antibody is usually conjugated to an enzyme or fluorophore for visualization.
Detection: The bound antibodies are visualized using chromogenic substrates or fluorescent compounds.
Chromogenic Detection: This method uses enzyme-conjugated secondary antibodies, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), which catalyze colorimetric reactions to produce a visible precipitate.
Fluorescent Detection: Secondary antibodies conjugated to fluorophores emit light when excited by specific wavelengths, allowing the use of fluorescence microscopy for visualization.
Cancer Diagnosis: IHC is extensively used to identify and classify different types of
cancers by detecting specific tumor markers.
Neuroscience: It helps in studying the distribution of
neurotransmitters, receptors, and other proteins in the nervous system.
Pathogen Detection: IHC can identify the presence of specific pathogens, such as viruses and bacteria, within tissue samples.
Developmental Biology: Researchers use IHC to examine the expression of developmental markers during embryogenesis and tissue differentiation.
Specificity: Using highly specific antibodies allows for precise localization of target antigens.
Versatility: IHC can be applied to a wide range of tissues and conditions.
Quantitative Analysis: Image analysis software can quantify staining intensity and distribution.
Limitations:
Antibody Quality: The success of IHC depends heavily on the quality and specificity of the antibodies used.
