To analyze the relationship between ROS and Smed-egfr-3 relative to ERK activation in early regeneration, a pERK immunostaining was performed after interfering with the aforesaid. Planarians were fixed at 6 and/or 24 h of regeneration and processed as previously described26. Bleached animals were washed with PBSTx (1 × PBS (10 × PBS: 1.37 M NaCl, 27 mM KCl, 100 mM Na2HPO4, 20 mM KH2PO4 in ultrapure H2O), 0.3% (v/v) Triton X-100) and incubated for 4 h in 1% blocking solution (1% (w/v) BSA in PBSTx) followed by the primary antibody (anti-pERK) diluted 1/1000 in blocking solution) overnight at 4 °C.

The immunostainings with anti-phospho-histone 3, anti-SYNAPSIN and anti-arrestin VC1 were carried out as described previously72. We used anti-phospho-histone3 (PH3, Cell signalling technology) to detect mitotic cells (diluted 1/300); anti-SYNAPSIN used as a pan-neural marker (diluted 1/50, Developmental Studies Hybridoma Bank)73; and, anti-VC-1, arrestin (VC-1, a mouse antibody specific for planarian photosensitive cells (1:15,000)74. After PBSTx washes and 1 h in blocking solution, they were incubated with the secondary antibody (goat anti-rabbit-POD diluted 1/500 in blocking solution) overnight at 4 °C. After PBSTx washes, samples were incubated for 8 min in TSA Plus Fluorescein solution (1/50 TSA Plus Fluorescein in 1 × Amplification Buffer (Tyramide Signal Amplification Labeling Kit No. 2; Molecular Probes, Thermo Fisher Scientific) in darkness. Samples were mounted after the final PBSTx washes (RT) and analysed with a MZ16F fluorescence stereomicroscope (Leica) equipped with a ProgRes C3 camera (Jenoptik, Jena, Germany).

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