*2.5. Fluorescence Quantification and Visualization*

Synchronized L1 larvae expressing an inducible green fluorescent protein (GFP) reporter for *gst-4, hsp-16.2, hsp-70, sod-3*, and *daf-16* genes were grown on NMG plates in the presence or absence of Q until the day of the assay, when they were subjected to or not subjected to thermally-induced oxidative stress (35 ◦C, 1 h). The precise day of assay was defined when a higher intensity of the fluorescence was observed after carrying out a screening with the different strains throughout the life of the worm, namely, day 3 in *gst-4* and day 5 in *hsp-70*. For the remaining strains, as no clear increase in the fluorescence was observed, the assessment was made in young (day 2 of adulthood) and older adult worms (day 9 of adulthood). In the cases of *hsp-16.2* and *hsp-70* reporter strains, after thermal stress, the worms were allowed to recover at 20 ◦C for 2 or 3 h respectively, before pictures were taken. The expression of *gst-4, hsp-16.2, hsp-70*, and *sod-3* was measured by quantifying the fluorescence of the GFP reporter. To analyze the subcellular localization of the DAF-16::GFP reporter, worms were classified as diffuse cytoplasmic, intermediate cytoplasmic/nuclear, and strong nuclear translocation. Approximately 35 randomly selected worms for each experiment were mounted in a 5 μL drop of 10 mM levamisole (except for DAF-16::GFP in 2% sodium azide) on a 3% agarose pad covered with a coverslip. All fluorescence determinations were done in an Olympus BX61 fluorescence microscope equipped with a filter set (excitation 470 ± 20 mn, emission 500 ± 20 nm) and a DP72 digital camera coupled to CellSens Software for image acquisition and analysis. ImageJ software was used to quantify fluorescence intensity. Three independent experiments were performed per assay and reporter strain.
