H2O2 plays many roles in cellular physiology. Therefore, we need tools for quantitative detection of H2O2 in tissues and whole model organisms. We recently introduced a genetically encoded H2O2 sensor, roGFP2-Orp1, which couples the redox-sensitive green fluorescent protein 2 (roGFP2) to the yeast H2O2 sensor protein Orp1. Expression of cytosolic or mitochondrial roGFP2-Orp1 in Drosophila allows the measurement of physiologically relevant changes in H2O2 levels, with compartment-specific resolution. Here, we provide a detailed protocol for the relative quantitation of H2O2 levels in living larvae by real-time imaging. We also describe a dissection and fixation method that conserves the redox state of the probe and thus allows reliable measurements on fixed adult tissues. Finally, we give recommendations for image processing, analysis, and interpretation, highlighting issues that require attention to detail, to ensure accuracy and validity of results.
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