Immunofluorescence: Dyes and Other Haptens Conjugated with Antibodies

Abstract Conjugation is the covalent attachment of a reporter molecule to a probe for the investigation of specific tissue constituents. Biotin, digoxigenin, fluorescent molecules (fluorochromes) and other haptens are applied for conjugation to antibodies and lectins. They enable the sensitive detection of a large variety of probes in cells and tissues by hapten–antihapten techniques. Fluorescently labelled probes are a prerequisite for the direct visualization of relevant markers not only in fixed tissues, but also in vivo and in vitro . The concomitant use of differently haptenylated antibodies facilitates bioanalytical approaches such as flow cytometry. Antibodies raised in the same animal species conjugated to various haptens are useful tools for specific multiple fluorescence labelling. Investigating pathologically altered tissues, haptenylated reagents allow for the omission of secondary antibodies avoiding undesired cross‐reactions with endogenous immunoglobulins in the tissues. Key concepts The fluorescence monitoring of biomolecules is enabled by chemically and genetically fluorochromated antibodies. Large biomolecules can be modified by fluorescent haptens or by nonfluorescent haptens to be targeted with fluorescent hapten‐binding molecules. Digoxigenylated, biotinylated and fluorochromated antibodies are useful tools for multiple labelling of antigens under physiological conditions and after pathological alterations, for example, in Alzheimer disease. Differently haptenylated antibodies allow for the immunofluorescence and immunoperoxidase staining with antibodies from the same host species and without possibly cross‐reacting secondary antibodies. Fluorescent primary antibodies are useful for the direct in vivo labelling. Alternative approaches include the combined immuno‐ and lectin‐histochemical staining of neural markers.