How does immunofluorescence function?
Any immunofluorescence (IF) technique will necessitate the employment of a fluorescent-labeled antibody to identify and localize proteins, antigens, or other biological molecules present within or outside the cell. The non-covalent binding of antigenic determinants occurs during the interaction of these purified antibodies, which might be monoclonal or polyclonal.
To visualize antibody binding to antigens, a specific fluorochrome, also known as a fluorescent dye, with different excitation and emission wavelength peaks is chosen based on the target molecule. For example, 4',6-diamidino-2-phenylindole (DAPI), which binds to A-T rich sections of the DNA double helix, is commonly used in immunofluorescence imaging of deoxyribonucleic acid (DNA).
Techniques of immunofluorescence
Direct immunofluorescence (IF), indirect IF, indirect IF complement-fixation (IF-CF), and double IF are the four major types of immunofluorescence methods.
Direct immunofluorescence
Direct immunofluorescence staining (IF) is a single-step histological staining process that enables for the observation of a single in vivo fluorescent-labeled antibody directed against a target antigen of interest. A typical direct IF approach for frozen tissue sections will begin with washing the sections in PBS, followed by incubation with fluorescent-tagged antibodies directed against the target antigen.
Direct IF antibodies typically exhibit specificities against IgG, IgM, IgA, complement 3 (C3), and fibrin. Notably, earlier research that confirmed the highest signal-to-background ratio set the concentration of each antibody.
Following incubation, the slides are washed and mounted for imaging with a fluorescence microscope. Direct IF has several advantages, including high specificity and low time requirements; nevertheless, the restricted number of antibodies available to bind to specific targets can limit the sensitivity of this approach in some applications.
Indirect immunofluorescence
In contrast to the one-step technique used for direct IF, indirect IF uses a two-step procedure. Instead of using a single fluorescently tagged antibody, indirect IF incubates the sample with an unlabeled primary antibody, followed by an incubation with a fluorophore-labeled secondary antibody directed against the Fc region of the primary antibody.
Although indirect IF takes longer, it is thought to be a far more sensitive technique than direct IF because many secondary antibodies can be used to bind to each primary antibody, thus increasing the potential fluorescence signal.
Although indirect IF has advantages such as improved sensitivity and the ability to utilize different conjugated secondary antibodies, it can also be more complex, especially in multiplex investigations combining several secondary antibodies from different target species.
Indirect IF-CF
Indirect IF-CF is a third type of immunofluorescence method. The essential working principle of indirect IF-CF is that the binding interaction between antibodies and their antigens generates a large number of C3 molecules. In comparison to indirect IF alone, indirect IF-CF is regarded as a far more sensitive amplification technique.
Double immunofluorescence
Double IF is the fourth and final form of IF method. Double IF allows for the identification and tagging of two distinct antigens present on a cell when utilized in in vitro investigations.
In vivo research using this imaging approach in either humans or animal models can distinguish between two antibodies tagged with distinct fluorophores.
Although double IF can be performed in either a direct or indirect manner, the indirect method is thought to be much more sensitive than the direct method.
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