Fluorescence is the property of absorbing light rays of one particular wavelength and emitting rays with a different wavelength. Fluorescent dyes show up brightly under ultraviolet light as they convert ultraviolet into visible light. Coons and his colleagues (1942) showed that fluorescent dyes can be conjugated to antibodies and that such labeled antibodies can be used to locate and identify antigens in tissue. Because of this reason, immunofluorescence technique is also a type of immunohistochemistry assay.
The most commonly used fluorescent dyes are fluorescein and rhodamine but other highly fluorescent substances such as phycoerythrin and phycobiliproteins have also been used. Dyes can be conjugated to the Fc region of an antibody molecule without affecting the specificity of antibody. Fluorescein, an organic dye that is the most widely used label for immunofluorescence procedures absorb blue light (490nm) and emits an intense yellow green fluorescence (517nm). Rhodamine, another organic dye absorb in the yellow green range (515nm) and emits a deep red fluorescence (546nm).
Fluorescent-antibody staining of cell membrane molecules or tissue sections can be direct or indirect. In direct staining, the specific antibody (the primary antibody) is directly conjugated with fluorescein; in indirect staining, the primary antibody is unlabeled and is detected with an additional fluorochrome-labeled reagent. A number of reagents have been developed for indirect staining. The most common is a fluorochrome-Labeled secondary antibody raised in one species against antibodies of another species, such as fluorescein-labeled goat anti-mouse immunoglobulin.
Fluorescence is the property of absorbing light rays of one particular wavelength and emitting rays with a different wavelength. Fluorescent dyes show up brightly under ultraviolet light as they convert ultraviolet into visible light. Coons and his colleagues (1942) showed that fluorescent dyes can be conjugated to antibodies and that such labeled antibodies can be used to locate and identify antigens in tissue. Because of this reason, immunofluorescence technique is also a type of immunohistochemistry assay. The most commonly used fluorescent dyes are fluorescein and rhodamine but other highly fluorescent substances such as phycoerythrin and phycobiliproteins have also been used. Dyes can be conjugated to the Fc region of an antibody molecule without affecting the specificity of antibody. Fluorescein, an organic dye that is the most widely used label for immunofluorescence procedures absorb blue light (490nm) and emits an intense yellow green fluorescence (517nm). Rhodamine, another organic dye absorb in the yellow green range (515nm) and emits a deep red fluorescence (546nm). Fluorescent-antibody staining of cell membrane molecules or tissue sections can be direct or indirect. In direct staining, the specific antibody (the primary antibody) is directly conjugated with fluorescein; in indirect staining, the primary antibody is unlabeled and is detected with an additional fluorochrome-labeled reagent. A number of reagents have been developed for indirect staining. The most common is a fluorochrome-Labeled secondary antibody raised in one species against antibodies of another species, such as fluorescein-labeled goat anti-mouse immunoglobulin.
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