Fluorescence detection Immunofluorescence

Immunofluorescence staining permits detection of protein antigens in situ, in order to investigate the subcellular localization or cellular distribution within a tissue. The cells or tissue sections are fixed and incubated with the specific primary monoclonal antibody. The antigen-primaiy monoclonal antibody complex is bound by a second antibody conjugated to a fluorescent dye, such as rhodamine-p-isothiocyanate or fluorescein isothiocyanate, for detection by fluorescence microscopy. Immunofluorescence staining is described in more detail in Chapter 16. 

Indirect immunofluorescence assay (IFA) A laboratory test used to detect antibodies in serum or other body fluid. The specific antibodies are labeled with a compound that will make them glow a fluorescent green color when observed microscopically under ultraviolet light. 

Bakkus, M.H., Brakel-van Peer, K.M., Adriaansem, H.J., Wierenga-Wolf, A.F., van den Akker, T.W., Dicke-Evingep M.J., and Benner, R. (1989) Detection of oncogene expression by fluorescent in situ hybridization in combination with immunofluorescent staining of cell surface markers. Oncogene 4,1255-1262. 

Controls have to be carried out incubation (and development without incubation with the primary antibody, i.e. only with secondary antibody) to control for nonspecific reactions of the second antibody. In addition, if immunofluorescence is used, unstained slides have to be investigated also as controls in order to detect nonspecific background fluorescence (for example of connective tissue), which may in some cases be enhanced by some drugs which were administered prior to taking the sample. 

In 1942 Coons et al. described an immunofluorescence technique for detecting cellular antigens in tissue sections [15].This method utilized a fluorescent label bound to the primary antibody to localize the target antigen. In this case the fluorescent label was the detection method. The use of an immunoenzyme approach to detect binding of the primary antibody was introduced a quarter of a century later [16] with the introduction of peroxidase-labeled antibodies. 

Figure 10.16. Endocytosis of dopamine reuptake transporters triggered by amphetamine, a Application of amphetamine reduces the transmembrane current that is a measure of dopamine reuptake. Shown are several independent experiments, b Detection of the transporter by immunofluorescence and confocal microscopy. In the absence of amphetamine, the transporter is located at the cell surfaces. Amphetamine induces translocation of the fluorescence to intracellular compartments.

Mixing of colors is less easily detected with enzyme methods than with double immunofluorescent labeling where filter systems in the microscope are more effective in segregating the staining intensities of the two labels. The use of fluorescent antibodies and enzyme-labeled antibodies for the different epitopes may lend itself particularly well to assess co-distribution of epitopes. 

Taxonomic probes, including antibodies and nucleic acid or peptide nucleic acid (PNA) probes selectively stain particular target cells by association with antigens or DNA/RNA. The corresponding approaches are designated as immunofluorescence (IF) and fluorescence in situ hybridisation (FISH), respectively. The major limitation of these labelling procedures is the low fluorescence intensity, which results in poorly labelled cells that escape detection. 

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