Fluorescence intensity measurements of general ROS-, H2O2- or pERK signal

The relative fluorescence intensities of samples with an in vivo general ROS or H₂O₂ staining as well as samples with a pERK immunostaining, were quantified using ImageJ (version 1.48v) on images taken with a MZ16F fluorescence stereomicroscope (Leica) equipped with a ProgRes C3 camera (Jenoptik, Jena, Germany). The mean intensity values were obtained for anterior and/or posterior blastema regions in case of the pERK immunostaining (FI-Blastema). When carried out an in vivo ROS/H₂O₂ stain, the mean intensity values were obtained from the H- and R-wound region (FI-Wound). In both cases, the background mean pixel intensity values were obtained (FI-Background; average of 3 distinct regions in the rest of the fragment, except the region at the ventral nerve cords and photoreceptors as there is a stronger pERK specific signal). The fluorescence intensity was expressed as the mean FI-Blastema or FI-Wound divided by the mean FI-Background, representing how many times the signal in the blastema or wound region is higher compared with the background.