Cloning of the eaat2 genes into the TOPO pCRII vector (TA Cloning Kit Dual Promoter, Invitrogen) is described elsewhere (Gesemann et al., 2010a (link)). Plasmids containing the genes were linearized for SP6 and T7 in vitro transcription and purified with phenol-chloroform. Digoxigenin (DIG)-labeled antisense RNA probes were generated using DIG-RNA-labeling kit (Roche Diagnostics). Larval whole-mount and adult retina in situ hybridization was done on 5-d postfertilization (5-dpf) larvae and adult retinal cross sections. Detailed protocol of in situ hybridization is described by Huang et al. (2012) (link). Briefly, the tissue was treated with proteinase K and postfixed with 4% paraformaldehyde (PFA) before prehybridization at 64°C. Hybridization of RNA probes was done at 64°C overnight. On day 2, after several stringency washes at 64°C, probes were blocked in 1× Roche blocking solution in Tris/NaCl/Tween. Anti-DIG AP antibody was applied overnight at 4°C. On day 3, after several washing steps, signal was detected by incubation in staining buffer. Stained embryos/retinal sections were fixed with PFA and imaged in glycerol (whole-mount) with an Olympus BX61 light microscope. Images were processed and assembled using Adobe Photoshop and Adobe Illustrator CS5.
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