Chapter 12 Nonfluorescent Immunolocalization of Antigens in Mitotic Sea Urchin Blastomeres

Abstract

Publisher Summary This chapter discusses the rationale, methodology, and some results of applying peroxidase–anti-peroxidase (PAP) staining for immunolocalization of antigens in early sea urchin blastomeres. Sea urchin eggs and early embryos have proved to be a useful system for biochemical and cytological analysis of the processes of cell division and embryonic development. The large volumes of gametes routinely obtained produce sufficient quantities of rapidly and synchronously dividing embryos for stage specific isolation, biochemical analysis, and immunolocalization of cellular proteins. The chapter considers this system to investigate the role of cytoskeletal motors, particularly the kinesin superfamily of MT-based motors, in dividing sea urchin blastomeres. Immunoperoxidase staining of sea urchin embryos with a tertiary PAP system avoids a number of the problems associated with immunofluorescence. The peroxidase reaction produces a colored product that is resistant to photobleaching. This stain is visible by simple bright-field illumination, thus decreasing background by avoiding cell autofluorescence and increasing specific information by utilizing a greater depth of field. The detection of a weak signal is also enhanced by tertiary labeling—using three sequential antibody probes, each of which geometrically increases specific signal, and a final signal amplification produced by a controllable, chromogenic, and enzymatic reaction to generate the desired degree of staining instead of relying on the amount of fluorescent stain conjugated to an antibody.