Validating Zebrafish Gnrh3 Antibody Specificity

The specificity of the zebrafish Gnrh3 Gap antibody was verified by confirming the recognition and specificity of the antibody to Gnrh3 expressed in a heterologous cell line and by co-localizing Gnrh3 immunoreactivity in gnrh3 transgenic adult brain sections (as described in next section), expressing tdTomato in gnrh3 neurons [13 (link)]. The entire zebrafish gnrh3 coding region (from 28 bp to 312 bp of NCBI Reference Sequence NM_182887.2) was cloned into the pcDNA3.1 (Life Technologies) mammalian expression vector under the control of the CMV promoter. The zebrafish gnrh3-pcDNA3.1 plasmid and the control pcDNA3.1 plasmid were transfected into COS7 cells with FuGENE 6.0 (Promega). As an additional control to check for cross-reactivity, we also transfected cells with the zebrafish gnrh2-pcDNA3.1 plasmid. The cells were grown in 25 cm² sterile cell culture flasks in DMEM supplemented with 10% FBS, 1% glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin (Biological Industries) at 37°C and 5% CO₂. After 48 hours of incubation, cells were transferred to chamber slides. After 24 hours, the cells were fixed with 4% paraformaldehyde (PFA) in PBS for 1 hour at room temperature and were washed with PBS. Blocking and immunostaining for zebrafish Gnrh3 Gap was then conducted as described in the next section.