Fig. 2. In vivo incorporation of tyramide-conjugates identifies endogenous ovoperoxidase activity. (A) The chemistry of peroxidase-mediated formation of dityrosine crosslinks between adjacent proteins (R¹, R²). This chemistry can be exploited to permanently bind a fluorochrome to proteins, as per the tyramide signal amplification mechanism (Bobrow et al., 1989). (B) The tyramide-fluorochrome conjugates compete with the formation of endogenous dityrosine crosslinks. (C) Eggs pretreated with the ovoperoxidase inhibitor 3-aminotriazole (3-AT; right) (Showman and Foerder, 1979) show significantly less tyramide-Alexa Fluor 488 incorporation in the fertilization envelope than untreated controls (left). Fluorescence images (top) are complemented by DIC images (bottom). (C') Fluorescence images from C overlaid on respective DIC images to show selectivity of incorporation. Scale bar: 100 µm. (D) Quantification of fluorescence intensity between control and 3-AT treated eggs. Mean fluorescence per unit area (AU, arbitrary units) at the equator of the fertilization envelope of each species. Standard deviation per treatment is shown. The number of replicates measured per treatment is indicated.