Thesis

Immunofluorescence localization of extracellular matrix component (s) in two species of sea urchin

An indirect immunofluorescence method was used to localize and trace the expression of an aggregation-promoting factor(s) (S-2) in the early stage embryos (1, 2, 4, 8, and 16-cell) of two species of sea urchin, Strongylocentrotus purpuratus and Lytechinus pictus. The S-2 antigen(s) was isolated from the supernatant of calcium-magnesium-free sea water dissociated S. purpuratus blastulae. Whole embryos from which fertilization membranes had been removed were incubated with purified anti-S-2 antibody (AS-2). Bound AS-2 was labeled with a second antibody (goat anti-rabbit IgG) that had been conjugated with fluorescein isothiocyanate. Immunofluorescence was assessed and photographed using a fluorescence microscope. The fertilized eggs and early stage embryos of both species of urchin displayed peripheral fluorescence. The antibody did not bind to the surfaces of unfertilized eggs nor did it penetrate the hardened fertilization membrane of embryos. Embryos incubated with preimmune rabbit serum showed no staining. (See more in text.)

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