Analysis of Sea Urchin Embryo Gene Expression by Immunocytochemistry

Abstract

Publisher Summary The technique of immunocytochemistry permits the detection of virtually any antigenic protein in cells and tissues. Immunocytochemistry involves specific antigen–antibody interactions and enables demonstration of not only the presence but also the intracellular localization of an antigen. It is also used simultaneously to detect multiple antigens, thus allowing comparison of their relative distributions. It has advantages over traditionally used histological and enzyme staining techniques. Immunocytochemistry is widely used to identify embryonic territories and specific cell lineages during normal sea urchin development. Several different fixation protocols are used successfully to localize a variety of antigens in sea urchin embryos. Fixation is the most important step in the entire immunocytochemical procedure. Permeabilization is essential if intracellular antigens are to be detected or if antigens are localized deep within the embryo. As older sea urchin larvae are large and fragile, special handling is required to maintain their integrity during fixation and processing for immunostaining. Several different lineage-specific monoclonal antibodies are currently available from various sea urchin labs and the Developmental Studies Hybridoma Bank. A wide variety of polyclonal antibodies are raised to sea urchin antigens. Indirect immunofluorescence is the most common detection method used for whole mount staining of sea urchin embryos. The large size and thickness of sea urchin larvae make them ideally suited for the confocal microscopic analysis of fluorescence immunostaining. Phalloidin staining alone or coupled with antibody staining is used to view the basic shape and developmental stage of the larvae. Fluorescently labeled phalloidin is used to visualize actin microfilaments particularly well in late sea urchin gastrulae and larvae. This allows the visualization of muscle at the prism stage and the developing adult rudiment in the late larva. The chapter describes more generally applicable problems that may be encountered whenever performing immunostaining.