Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on biological samples. This technique uses the specificity of antibodies for their antigen while using fluorescent dyes to generate a signal of the antigen antibody within a cell, and therefore allows visualization of the distribution of the target molecule through the sample.
Direct immunofluorescence uses a single, primary antibody, chemically linked to a fluorophore or fluorochrome. The primary antibody recognizes the target molecule (antigen) and binds to a specific region called the epitope. The attached fluorophore can be detected via fluorescent microscopy, which, depending on the fluorophore used, will emit a specific wavelength of light once excited. Direct immunofluorescence, although somewhat less common, has notable advantages over the indirect procedure. The direct detection of the antigen by a primary antibody directly labeled with a fluorochrome reduces the number of steps in the procedure, saving time and reducing non-specific background signal.
Indirect immunofluorescence uses two antibodies; the unlabeled first (primary) antibody specifically binds the target antigen, and the secondary antibody, which is labeled with a fluorophore, recognizes the primary antibody and binds to it. Multiple secondary antibodies can bind a single primary antibody. This provides signal amplification by increasing the number of fluorophore molecules per antigen. This protocol is more complex and time-consuming than the primary (or direct) protocol above, but allows more flexibility because a variety of different secondary antibodies and detection techniques can be used for a given primary antibody.