Labeling and use of monoclonal antibodies in immunofluorescence: Protocols for cytoskeletal and nuclear antigens

Abstract

Antibodies are widely used to target and label specifically extra- or intracellular antigens within cells and tissues. Most protocols follow an indirect approach implying the successive incubation with primary and secondary antibodies. In these protocols the primary antibodies are specifically targeted against the antigen in question and are normally not labeled. The secondary antibodies come from a different species and are in contrast fluorescently labeled. The idea is that the primary antibodies specifically bind to their targets but cannot be visualized directly. Only binding of the secondary (fluorescent) antibodies to the constant region of the primary antibodies allows consecutively the visualization in a fluorescent microscope. Primary antibodies can be either of monoclonal (normally produced in mouse) or of polyclonal origin (normally produced in rabbit, goat, sheep, or donkey). Using (primary) monoclonal antibodies has the clear advantage that all antibodies used are identical in origin and behavior and should thus give a more clear-cut labeling result. On the other hand the demands towards labeling protocols might be concomitantly higher: Binding of primary antibodies will only occur if fixation and labeling protocols preserve the antigen sufficiently to keep its specific and unique target structure available. One could imagine that for polyclonal antibodies this demand is slightly lower as there is a pool of antibodies with varying specificities against multiple parts of their target antigens. Certain fractions of this pool might thus tolerate a larger variety of conditions, and consequently a larger variety of protocols might still result in successful labeling. Each step in a labeling protocol can be decisive for the outcome of an experiment especially if monoclonal antibodies are used. Especially critical are choice of buffer and fixation and permeabilization parameters of the protocol. In this chapter we discuss and detail proven protocols using monoclonal antibodies for two key targets within a cell, namely, (a) the cytoskeleton with emphasis on microtubules (Cramer and Mitchison, J Cell Biol 31:179-189, 1995; Traub et al., Biol Cell 90:319-337, 1998; Desai et al., Methods Cell Biol 61:385-412, 1999) and (b) the nuclear pore complex (Davis and Blobel, Cell 45:699-709, 1986; Kimura et al., Mol Cell Biol 23:1304-1315, 2003). The two protocols which differ substantially in using either a chemical fixation/permeabilization approach versus a one-step coagulation protocol are chosen to offer the reader tools to design their own procedures and adapt them accordingly to fit their individual experimental setup and the biological question asked. © 2014 Springer Science+Business Media, New York.