Gnrh elisa protocol

ME explants were dissected from female rats (3–4 months old, n=18) and processed as previously described32 (link). Briefly, ME explants were incubated in artificial cerebrospinal fluid with the following composition (in mM): NaCl 117, KCl 4.7, NaH₂PO₄ 1.2, NaHCO₃ 25, CaCl₂ 2.5, MgCl₂ 1.2, glucose 10, bubbled with 95% O₂–5% CO₂ (pH 7.4, osmolarity 304 mOsm). Medium was collected before and after explants were treated with or without recombinant human Semaphorin-7A/Fc chimera (1 μg ml⁻¹; 1835-S3, R&D Systems), followed by incubation at 37 °C for 4 h at 300 r.p.m. Explants were treated with KCl 0.05 M at the end of the incubation period to confirm their viability. Collected media were analysed for GnRH content following a GnRH ELISA protocol (Phoenix Pharmaceuticals Inc., California, catalogue number FEK-040-02).

Adult female rats (4-month old) with regular 4-day oestrous cycle were killed on the day of dioestrus and ME explants were dissected and processed as follows: ME explants were incubated in artificial cerebrospinal fluid (aCSF) with the following composition (in mM): NaCl, 117; KCl, 4.7; NaH₂PO₄, 1.2; NaHCO₃, 25; CaCl₂, 2.5; MgCl₂, 1.2; glucose, 10, bubbled with 95% O₂-5% CO₂. (pH 7.4, osmolarity 304 mOsm). Medium was collected before and after explants were treated with recombinant human anti-Müllerian Hormone (AMH, R&D Systems, 125 nM), recombinant human TGFβ-1 (Millipore, 2 nM) or both, followed by incubation at 37 °C for 4 h at 300 r.p.m. The same concentration of TGFβ-1 has previously been used to evaluate GnRH secretion from ME explants33 (link). Explants were treated with 0.05 M KCl at the end of the incubation period to confirm their viability. Collected media were analysed for GnRH content following a GnRH ELISA protocol (Phoenix Pharmaceuticals Inc., California, Catalog no. # FEK-040-02).

Adult female mice were sacrificed on the day of diestrus and ME explants were dissected and processed as previously described (Prevot et al, 1999). Conditioned medium was collected after explants were treated with increasing doses of rFGF21 (20 min) or with KCl 0.05 M in order to induce the release of the entire GnRH vesicle pool. Collected media were analyzed for GnRH content following a GnRH ELISA protocol (Phoenix Pharmaceuticals Inc., California, Catalog no. #FEK‐040‐02).