Alexa 555 labelled goat anti rabbit igg

Two of the three series of brain sections from each fish were used for PCNA immunohistochemistry, while the other series was used for GnRH3 labelling to confirm the location of the TN. Immunohistochemistry with an anti-GnRH3 antibody was performed using a custom-made primary anti-GnRH3 antibody (produced by Protein Purify, Isezaki, Japan) and Alexa 555-labelled goat anti-rabbit IgG (ThermoFisher Scientific LTD)^{16 (link)}. GnRH3-positive neurons were observed under a fluorescence microscope (Axio Imager A1, Zeiss, Jena, Germany) and sections with the highest number of GnRH3-positive cells were identified.

The brain slices at 72 h after cultivation were fixed with 4% paraformaldehyde. They were washed gently with PBS three times. Immunocytochemistry with an anti-GnRH3 antibody was performed using a custom-made rabbit anti-GnRH3 antibody (×7000, produced by Protein Purify, Isesaki, Japan) and Alexa 555-labelled goat anti-rabbit IgG (×500, Thermo Fisher Scientific, Ltd.). For nuclear staining, Hoechst 33342 solution (1000×, Wako Pure Chemical Co.) was used. After staining, coverslips were mounted with ProLong Gold antifade reagent (Thermo Fisher Scientific, Ltd.). The stained slices were observed using a confocal laser microscope (LSM 5 PASCAL, Carl-Zeiss AG, Jena, Germany). Since GnRH3 neurons are located so close to each other that it is difficult to count them accurately on a single sectional image. Then, we obtained a series of images in the z-stack and merged them to count the actual number of GnRH3 neurons in each slice. The total number of GnRH3 neurons per animal was calculated by summing the number of GnRH3 neurons on each half side of the brain slices (2–4 slices) originating from the same animal.