At 4 dpf, healthy larvae were injected at the pericardial site with PEG–PAMAM–Rho solutions of 5, 20, and 50 mg/mL (freshly prepared in 0.9% NaCl saline solution). As negative controls, non-injected animals and animals injected with vehicle (0.9% NaCl saline solution) were used. Larvae were anesthetized with 140 mg/L tricaine (MS222; ethyl-3-aminobenzoate methanesulfonate, #E10521, Sigma-Aldrich) [26 (link)] in E3 medium with PTU and positioned in custom-design channels molded in 3% (w/v) agarose in E3 medium (see details in Figure S1, Supplementary Material ). Injection was performed by loading injection solutions in borosilicate glass capillaries (GB100F-10P, Science Products GmbH, Hofheim am Taunus, Germany). Capillaries were attached to the micromanipulator (M-152, Narishige, Setagaya, Japan) and injection controlled by a microinjector (IM 300 microinjector, Narishige, Setagaya, Japan). Injection pressure was adjusted to inject 2–5 nL of the tested solutions at the pericardial site. After injection, larvae were transferred to Petri dishes containing fresh E3 with or without PTU medium (for imaging or for behavioral tests, respectively) and incubated at 28 ± 0.5 °C until further analysis.
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