The production of antibodies in vitro was introduced by Georges Köhler and César Milstein, who first employed cultures of fused cells to isolate antibodies of defined specificity. In this procedure, termed monoclonal antibody production, antibodies are first produced in mice, and activated B-lymphocytes from the host's spleen (the source of antibodies) are fused with cultured plasma tumor myeloma cells (that do not produce antibodies) from mice of the same strain. The fusion product is a hybrid (termed a hybridoma) of the two cells that continues to grow and divide in culture and produces large amounts of the antibody. Unfused cells and non-productive fusions are eliminated by classical cloning techniques to purify the culture of antibody-producing cells. A hybrid cell line produces only a single type of antibody, which can be used as a selection marker in serial dilution cloning by monitoring the culture medium for secreted antibody at each stage. Ultimately a cell line is established from a single cell (monoclonal) in which all of the cells produce an identical antibody.

In a double immunofluorescence experiment illustrated above, a culture of Madin-Darby ovine kidney cells was fixed, permeabilized, and blocked with 10-percent goat serum before being treated with a cocktail of mouse anti-histones (pan) and rabbit anti-giantin primary antibodies. The target organelles were visualized with goat anti-mouse and anti-rabbit secondary antibodies (IgG) conjugated to Texas Red and Alexa Fluor 488, respectively. The filamentous actin cytoskeletal network was imaged 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.