Fig. 3. Kinetics of ovoperoxidase crosslinking is linked to hydrogen peroxide synthesis. The incorporation of fluorescent tyramide analogs can be visualized in real time, and is limited to the fertilization envelope. (A) Representative snapshots of Strongylocentrotus purpuratus fertilization at times shown, where sperm fusion (arrowhead) represents time=0 (seconds). The concentration of tyramide-Alexa Fluor 594 (red) within the fertilization envelope (arrowhead) increases over time. The plasma membrane is counter-stained with FM1-43 (green). Fluorescence images (top) are complemented by DIC images (bottom). Scale bar: 50 µm. (B) Quantification of tyramide-Alexa Fluor 594 incorporation into the fertilization envelope (red) versus hydrogen peroxide production over time (blue) (Wong et al., 2004). Mean accumulation of tyramide fluorescence in the fertilization envelope shown (solid line); squares represent individual data sets per egg (total shown, n=4). Inset: Rate of tyramide-Alexa Fluor 594 incorporation into the fertilization envelope, as a proxy for ovoperoxidase activity (red), versus rate of hydrogen peroxide production by Udx1 (blue) (Wong et al., 2004). Plots show the percentage of final fluorescence intensity (tyramide-Alexa Fluor 594) or the maximal rate of hydrogen peroxide synthesis.