Immunohistochemistry | Immunohistochemistry
Key Words: confocal, Fluorescence, fluorophore, immunocytochemistry
Definition:Immunohistochemistry exploits antigen-antibody interactions to locate specific proteins in tissues. The antibody is usually linked to other molecules to aid visualization (fluorophore, reporter enzyme, etc)
TECHNOLOGY:
In immunohistochemistry the specificity of the antibody-antigen interaction is used to selectively identify specific proteins in cells or tissue sections. It may be carried out on ruptured cells or on frozen or fixed tissue sections. In direct immunohistochemical staining one labeled antibody binds directly to its antigen. While this uses only one antibody and is simple and rapid to carry out, direct immunohistochemical staining may suffer from lack of sensitivity. It is more common to use indirect methods, where a second layer of labeled antibodies binds to the primary antibody bound to the antigen. Several secondary antibodies may be able to bind different antigenic sites on the primary antibody, therefore amplifying the signal to be detected. The antibody-antigen reaction may be visualized using a tertiary reagent such as a reporter enzyme (such as peroxidase that catalyses a color-forming reaction), colloidal metal, hapten, radiolabel or fluorophore (such as Texas Red or FITC, or especially in deep imaging, quantum dots). In the case of a reporter enzyme system, addition of a suitable substrate (chromogen) such as AEC (3-amino, 9-ethyl carbazol), or DAB (diaminobenzadine) results in the production of a colored product that stains the cell.
APPLICATIONS:
Immunohistochemistry is widely used in pathology and clinical diagnostics to detect disease markers, especially in cancer. It is also widely used in research applications for the localization of specific proteins.
MICROSCOPE CONFIGURATION:
Immunohistochemically stained tissue sections are most frequently viewed using brightfield techniques on an upright microscope. Fluorophores require epi-fluorescence and may be visualized using widefield or confocal techniques. Confocal imaging offers the greatest scope for spatial localization of proteins in thicker specimens.
RECOMMENDED SYSTEM:
Nikon's i-series upright clinical and research microscopes provide exceptional brightfield microscopy ideal for the examination of tissue sections. The Eclipse 55i, for example, incorporates a unique white LED illumination system ideal for bright field, while the 80i and 90i incorporate epifluorescence capability. For confocal applications, users can choose from the EC1 entry level instrument to the C1si spectral imaging system or LiveScan Sweptfield systems for the most sophisticated of research applications.
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