Following injury, the inflammatory response directs the host immune cells to the wound to maintain tissue integrity and combat pathogens. The recruitment of immune cells to inflammatory sites is achieved through the establishment of a variety of signal gradients. Using a zebrafish embryo injury model, it was recently demonstrated that, upon injury, cells at the wound margin rapidly produce hydrogen peroxide (H(2)O(2)) which serves as an early paracrine signal to leukocytes. This chapter provides a method for performing in vivo time-lapse fluorescence microscopy to visualize leukocyte behaviors and wound-produced H(2)O(2) simultaneously in single zebrafish embryos during an acute inflammatory response. Protocols are included for inducing a robust, reproducible acute inflammatory response, for rapidly mounting immobilized embryos for time-lapse imaging, and for computing ratiometric data from the images of embryos expressing the genetically encoded H(2)O(2) sensor fluorophore HyPer. General issues to consider when designing multichannel fluorescent imaging are discussed, including particular considerations to note when monitoring intracellular H(2)O(2) concentration dynamics using HyPer.
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