Antibodies are generated by immunizing susceptible hosts, such as rabbits, mice, goats, chickens, etc., with a specific antigen. If the antigen is derived from tissue, it must be exhaustively purified, but many antigens are now synthetic portions of a larger protein or carbohydrate. If a large protein is used as the antigen, such as a multi-subunit globular protein, the antibodies produced by the donor animal are not likely to be pure, but will usually be directed towards several portions (in effect, the subunits) of the large conglomerate. These antibodies are referred to as being polyclonal, and usually contain antibodies that react to carrier proteins and other native antibodies that may react to other tissue components. In this case, immunological reactions are usually not specific, targeting the desired antigen-antibody reaction, and are often complicated by high levels of background noise.

In a double immunofluorescence experiment, fixed and permeabilized adherent HJ1.Ov cells were treated with a cocktail of mouse anti-histones (pan) and rabbit anti-PMP 70 (peroxisomal membrane protein) primary antibodies, followed by a second mixture of goat anti-mouse and anti-rabbit secondary antibodies conjugated to Alexa Fluor 568 and Alexa Fluor 488, respectively (targeting the nucleus and peroxisomes). The filamentous actin cytoskeletal network in the cell culture illustrated above was stained with Alexa Fluor 350 conjugated to phalloidin. Images were recorded in grayscale with a QImaging Retiga Fast-EXi camera system coupled to an Olympus BX-51 microscope equipped with bandpass emission fluorescence filter optical blocks provided by Omega Optical. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles.