Cloning and Visualization of eaat2a in Zebrafish

eaat2a (ENSDARG00000102453) cloning into the TOPO pCRII vector (TA Cloning Kit Dual Promoter, Invitrogen, Basel, Switzerland) and preparation of digoxigenin (DIG)‐labeled antisense RNA probes is described elsewhere (Gesemann et al., 2010 (link); Niklaus et al., 2017 (link)). RNA probes were applied on whole‐mount zebrafish larvae (3 and 5 days post fertilization [dpf]) and adult (older than 6 months) brain cross sections at a concentration of 2 ng/μl at 64°C overnight (Huang et al., 2012 (link)). For larval brain sections, representative stained and paraformaldehyde (PFA) post‐fixed embryos were cryoprotected in 30% sucrose at 4°C overnight, embedded in Tissue Freezing Medium TFMTM (Electron Microscopy Sciences), cryo‐sectioned at 14–16 μm and mounted onto Superfrost slides (Thermo Fisher Scientific). PFA post‐fixed whole‐mount embryos (in glycerol) and sections were imaged with an Olympus BX61 brightfield microscope. Images were adjusted for brightness and contrast using Affinity Photo Version 1.8 and assembled in Affinity Designer Version 1.7.