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Reagents designed for artificial coloration of cells and/or tissues that have been labeled with a primary antibody during immunocytochemistry or immunohistochemistry tests. Typically, these reagents are intended to allow chromogenic or fluorescent detection of antibody binding. Chromogenic detection methods typically use a secondary antibody conjugated to an enzyme (e.g., alkaline phosphatase, horseradish peroxidase). When the enzyme is provided the appropriate substrate (e.g., nitro-blue tetrazolium and 5-bromo-4-chloro-3\'-indolyphosphate [BCIP/NBT], diaminobenzidine [DAB]), it will form colored precipitates that stain the cells. Slides stained using chromogenic methods are typically visualized using light microscopy. Fluorescent detection methods typically use a secondary antibody conjugated to a fluorophore (e.g., fluorescein isothiocyanate [FITC], rhodamine) that re-emits light at a specific wavelength when exposed to light. Slides stained with flourophores are typically visualized using fluorescence or confocal microscopy. Simultaneous staining of multiple primary antibodies can be accomplished by appropriate selection of secondary antibodies that are able to discriminate between the used primary antibodies. In the case of chromogenic detection methods, secondary antibodies conjugated to enzymes specific for different substrates and producing different colored pigments are used. In the case of fluorescent methods, secondary antibodies conjugated to fluorophores with non-overlapping excitation and emission spectra are used.
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