The idea for immunofluorescence originated with Albert H. Coons and his collaborators, who were the first to conjugate a fluorescent dye to an antibody and use it identify a tissue antigen with fluorescence microscopy. The dye used in the original conjugation reaction was fluorescein isocyanate, but this species has now largely been replaced with the isothiocyanate derivative (fluorescein isothiocyanate, FITC), which forms a more stable conjugate. In most cases, when fluorescein is conjugated to an antibody or antibody fragment, the molecule fluoresces with a bright apple-green color after absorbing blue light in the region of 490 nanometers. A wide spectrum of alternative chromophores with fluorescent colors covering the entire visible and near-infrared spectrum are now commercially available as antibody conjugates, including the Alexa Fluor dyes, cyanine dyes (Cy2, Cy3, Cy5, etc.), and many rhodamine derivatives.

A goat anti-mouse secondary antibody conjugate of Texas Red-X was employed to visualize mouse anti-alpha-tubulin (IgG) primary antibodies in the adherent culture of African water mongoose cells illustrated above. In addition, the cytoskeletal architecture was imaged with Alexa Fluor 488 conjugated to phalloidin, and DNA in the nucleus was targeted with the bis-benzimide fluorophore, Hoechst 33258. 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.