Immunohistochemistry techniques, strengths, limitations and applications

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Immunohistochemistry (IHC) is an important assessment tool that falls under immunostaining techniques and exploits the antigenantibody binding to study the status of target molecules in tissues of interest. IHC, in combination with microscopy and image analysis techniques, has become a very powerful tool that offers direct visualization of tissue antigens using antigen-specific labeled antibodies. Due to the versatility, simplicity and affordability of this technique, IHC is now indispensable in the fields of histology, pathology, cancer biology, neuroscience and drug discovery. .


Antibodies are small protein molecules that are naturally expressed by the body’s immune system in response to the entry of a foreign molecule (antigen) and help neutralize it. Thus, antibodies act as a defense against potentially dangerous foreign organisms and their products. The beauty of antigenantibody reaction is that each antibody is only specific for a part of the antigen, called an epitope, and does not bind other molecules that do not match its target, including the body’s own molecules. All immunostaining techniques, including IHC, use this important property of the antigenspecificity of the antibody reaction to ensure the detection of a single type of molecule from a medium of thousands of different molecules (Figure 1).


Figure 1: Schematic representation of antigen specificityantibody reaction that allows the detection and localization of a single target in a medium of thousands of intracellular molecules.


While the development of this important technique would not have been possible without the discovery of antibodies by Emil von Behring and Shibasabura Kitasato in 1890,1 it was not until 1923 that the antigenThe antibody complex was detected by Michael Heidelberger using labeled antigens.2 This was followed by the work of John Richardson Marrack describing the nature of the antigenantibody reaction. Marrack was the first to attach a dye to an antibody in 1934.3,4 The labeling of antibodies with the fluorescent tag, fluorescein, and the detection of their respective antigens in cells and tissues was pioneered by Albert Hewett Coons and others in 1941.5 and launched the immunostaining revolution.6 The technique has developed and improved greatly since then and has become an essential tool for discovery and diagnosis.

What is immunohistochemistry (IHC)?

IHC uses a combination of histology, anatomy, immunology, and biochemistry to detect the quantity, distribution, and location of a specific target in a tissue. Antibodies against the molecule of interest, often a protein, are generated in an organism of a different species and are usually labeled or aided by another set of labeled antibodies. Tissue samples are prepared using specialized techniques to allow entry of antibodies, and the label is detected using light or electron microscopy (Figure 2).

Immunohistochemical staining for tryptophan hydroxylase 2, revealing serotonergic neurons and their projections in human dorsal raphe.


Figure 2: Immunohistochemical staining for tryptophan hydroxylase 2, revealing serotonergic neurons and their projections in human dorsal raphe. Credit: Human Protein Atlasimage available at v21.1.proteinatlas.org.

What is the difference between immunostaining and immunohistochemical staining (IHC staining)?

Immunolabelling is a generic term that encompasses all the techniques used for the detection of molecules employing the antigenantibody reaction. Immunohistochemistry or immunohistochemical staining is a specific use case of immunostaining when the antigenantibody reaction is used to study the status of molecules in tissue (Greek historicalwhich means fabric).

What is the difference between immunohistochemistry and immunofluorescence?

The terms immunohistochemistry and immunofluorescence are applied depending on the standard sample and detection method used in the immunostaining technique. Immunohistochemistry refers to the evaluation of target antigens in fabrics when the detection method can be either chromogenic or fluorogenic. Immunofluorescence, on the other hand, can refer to both the evaluation of target antigens in cells and tissues and specifically involves the detection of a fluorescent label (fluorophore) (Figure 3).

Immunofluorescence for tyrosine hydroxylase revealing dopaminergic neurons in mouse midbrain.


Figure 3: Immunofluorescence for tyrosine hydroxylase revealing dopaminergic neurons in mouse midbrain. Credit: Human Protein Atlas, image available at v21.1.proteinatlas.org.

What is the difference between immunocytochemistry and immunohistochemistry?

While immunohistochemistry involves the study of the status of antigens in tissue samples, immunocytochemistry refers to immunostaining when the sample of interest is cells in culture (from the Greek cytomeaning cells).

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