Understand the difference between direct and indirect methods for immunofluorescence.
Immunofluorescence (IF) or cell imaging techniques rely on the use of antibodies to label a specific target antigen with a fluorescent dye (also called fluorophores or fluorochromes ) such as fluorescein isothiocyanate ( FITC ). Antibodies that are chemically conjugated to fluorophores are commonly used in IF. The fluorophore allows visualization of the target distribution in the sample under a fluorescent microscope ( eg epifluorescence and confocal microscopes). We distinguish between two IF methods depending on whether the fluorophore is conjugated to the primary or the secondary antibody: Direct IF uses a single antibody directed against the target of interest. The primary antibody is directly conjugated to a fluorophore. Indirect IF uses two antibodies. The primary antibody is unconjugated and a fluorophore-conjugated secondary antibody directed against the primar...