Anti kiss1r

The behavior of hfHypo cells was analyzed by Laser Scanning Confocal Microscopy (LSCM, Fluoview FV300, Olympus) after the immunostaining of neuronal markers (β-Tub III and Neuronal nuclei, NeuN), GnRH phenotype markers (GnRH peptide and kisspeptin receptor, KISS1R) and estrogenic receptors (ER β and G proteincoupled receptor 30, GPR30). Neurons were fixed in 4% (wt/vol) paraformaldehyde for 30 min, followed by permeabilization and blocking with a solution containing 0.3% (vol/vol) Triton X-100 and 10% (vol/vol) FBS in PBS for 1 h at 37 C. Samples were then incubated overnight at 4 C with the following primary antibodies: anti-βTub III (1:500, Covance), anti-NeuN (1:200, Millipore), anti-GnRH (1:100, Abcam), anti-KISS1R (1:200, Santa Cruz Biotechnology), anti-ERβ (1:200, Santa Cruz Biotechnology) and anti-GPR30 (1:200, Abcam). Secondary antibodies, Cy2 TM -conjugated Affini Pure donkey anti-rabbit IgG, Cy3 TM -conjugated Affini Pure donkey anti-mouseIgG and a Cy5 TM -conjugated Affini Pure donkey anti-goat IgG (1:500, Jackson ImmunoResearch Europe Ltd.) were then added for 1 h at RT. Finally, cells were counterstained with 200 ng/ml DAPI (Molecular Probes), for nuclear localization.