Tissue freezing medium tfmtm

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.

Generation of the chicken anti‐EAAT2a (zebrafish) antibody is described elsewhere (Niklaus et al., 2017 (link)). Five dpf larvae were fixed in 4% PFA in phosphate buffered saline (PBS, pH 7.4) at room temperature for 40 min. Embryos were cryo‐protected in 30% sucrose in PBS 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). Immunohistochemistry was performed as described before (Niklaus et al., 2017 (link)). Chicken anti‐EAAT2a 1:500, mouse anti‐synaptic vesicle 2 (IgG1, 1:100, DSHB USA), mouse anti‐acetylated tubulin (IgG2b, 1:500, Sigma 7451) and mouse anti–glutamine synthetase (IgG2a, 1:200, EMD Millipore, MAB302) were used as primary antibodies. Secondary antibodies were goat anti‐chicken Alexa Fluor 488, goat anti‐mouse IgG2a Alexa Fluor 568, goat anti‐mouse IgG2b Alexa Fluor 647 and goat anti‐mouse IgG1 Alexa Fluor 647, all 1:500 (all from Invitrogen, Thermo Fisher Scientific). Slides were cover‐slipped using Mowiol (Polysciences) containing DABCO (Sigma‐Aldrich) and imaged with a TCS LSI confocal microscope (Leica Microsystems). Images were adjusted for brightness and contrast using Affinity Photo Version 1.8 and assembled in Affinity Designer Version 1.7.