Primary neuron culture

Brains from 42 hpf zebrafish embryos were placed in 1x E2 medium (15 mM NaCl, 0.5 mM KCl, 1 mM MgSO₄, 150 μM KH₂PO₄, 50μM Na₂HPO₄, 1 mM CaCl₂, 0.7 mM NaHCO₃, 0.5 mg/l methylene blue) with 1x tricaine as described by Chen et al. [23]. Briefly, cells were grown in Dulbecco’s modified medium to 48 hpf. Morphine (1 nM) was added after plating. 48 hours later neurons were fixed in 4% paraformaldehyde (PFA) (w/v). Immunocytochemistry analysis for primary cultured cells was carried out as described by Chen et al. [23]. Neurons were fixed with 4% PFA (w/v) for 20 minutes and blocked with antibody buffer and 5% goat serum; 1:250 dilution was used for Oprm1 antibody. To-PRO (1:1000, ThermoFisher) and Alexa fluor 594 Phalloidin (1:10, ThermoFisher) were used as nuclei and soma markers, respectively. Primary cultures were analyzed with a life cell microscopy (Zeiss).