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 (H2O2) 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 H2O2 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 timelapse imaging, and for computing ratiometric data from the images of embryos expressing the genetically encoded H2O2 sensor fluorophore HyPer. General issues to consider when designing multichannel fluorescent imaging are discussed, including particular considerations to note when monitoring intracellular H2O2 concentration dynamics using HyPer.
|Title of host publication||Imaging and Spectroscopic Analysis of Living Cells: Imaging Live Cells in Health and Disease|
|Editors||P M Conn|
|Place of Publication||USA|
|Pages||135 - 156|
|Number of pages||22|
|Publication status||Published - 2012|
Pase, L., Nowell, C. J., & Lieschke, G. J. (2012). In vivo real-time visualization of leukocytes and intracellular hydrogen peroxide levels during a zebrafish acute inflammation assay. In P. M. Conn (Ed.), Imaging and Spectroscopic Analysis of Living Cells: Imaging Live Cells in Health and Disease (1st ed., pp. 135 - 156). Academic Press. https://doi.org/10.1016/B978-0-12-391856-7..00032-9