Anti erβ

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.

Slides prepared as described previously were washed three times with PBS, and non-specific binding sites were blocked with 5% BSA (w/v) in PBS for 4 h at room temperature in a humidity chamber. After three washes in PBS, spermatozoa were incubated overnight at 4°C with the primary antibody (anti-ERα (RRID:AB_640249), anti-ERβ (RRID:AB_2102246) and anti-PR (RRID:AB_632263) rabbit polyclonal antibody, respectively; Santa Cruz Biotechnology), diluted 1:50 (v/v) in PBS containing 1% BSA (w/v). After three washes in PBS, the cells were incubated with the secondary antibody (Alexa Fluor 488 chicken anti-rabbit; Thermo Fisher Scientific; Cat# A-21441, RRID:AB_2535859), diluted 1:800 (v/v), for 1.5 h at room temperature in a humidity chamber. The slides were then washed three times with PBS before the addition of 5 µL of 0.22 M triethylenediamine (DABCO) in glycerol:PBS (9 : 1 v/v) to enhance and preserve cell fluorescence. Finally, the preparations were covered with coverslips, sealed with colourless enamel and visualized using a Nikon Eclipse E-400 microscope under epifluorescent illumination. All samples were processed in duplicate and at least 150 spermatozoa were scored per slide.

RBCs were fixed with 3.7% formaldehyde in PBS (pH 7.4) for 10 min at room temperature, washed in the same buffer and permeabilized with 0.5% Triton X-100 in PBS for 5 min at room temperature. After washing with cold PBS, samples were incubated for 30 minutes at 37°C with monoclonal or polyclonal antibodies. Monoclonal antibodies: anti-ER-α, anti-ER-β, anti-ERK 1/2, anti-P38, anti-phosphorylated P38 (all Santa Cruz Biotechnology, Santa Cruz, CA) and anti-phosphorylated ERK 1/2 (BD PharMingen, San Diego, CA). Polyclonal antibodies: anti-AKT, anti-phosphorylated AKT (Thr 308)-R (all Santa Cruz Biotechnology), and anti-eNOS (phospho S-1177) (Cambridge, MA). As negative control we used mouse or rabbit IgG1 immunoglobulin isotype (Sigma). Samples were washed thrice in PBS to be then incubated with secondary antibody FITC-conjugated: anti-mouse (Invitrogen, Carlsbad, CA) or anti-rabbit (Invitrogen, Carlsbad, CA). For NO detection, samples were incubated with 4, 5-Diaminofluorescein diacetate (Enzo Life Sciences, Lausen, Switzerland) for 30 min at 37°C and washed three times in PBS. All the samples were recorded with a FACScan flow cytometer (Becton-Dickinson, Mountain View, CA, USA) equipped with a 488nm argon laser. At least 20, 000 events were acquired. The median values of fluorescence intensity histograms were used to provide a semi-quantitative analysis.