Oxytocin

Cultured N2a cells were transfected with pCS2 or pCS2-HAmOXTR. Live cells were then incubated with aCSF containing 1 mM oxytocin (GL Biochem) for 20 min, and then fixed in 4% PFA for 1 h. As control, cells were incubated in aCSF containing 1 mM Vasopressin (GL Biochem). Immunostaining was performed with the following antibodies: oxytocin (Phoenix Pharmaceuticals, Cat# G-051–01, RRID: AB_2876858; 1:250), HA (Covance, Cat# MMS-101P, RRID: AB_2314672; 1:200), Donkey anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific, Cat# A-21206, RRID:AB_2535792; 1:500) and Donkey anti-mouse Alexa Fluor 568 (Thermo Fisher Scientific, Cat# A10037; 1:500). For the antibody block experiment, the oxytocin antibody was pre-incubated with 10 mM oxytocin. Sections were incubated with 640/660 Deep-Red Fluorescent Nissl Stain (Thermo Fisher Scientific Cat# N21483) for 20 min at room temperature, washed, and mounted onto glass slides in 70% glycerol. Images were acquired on a Nikon A1 confocal microscope with a Plan Apo VC 20× DIC N2 (N.A. = 0.8) objective.

All blood tubes were centrifuged at 3,000 × g at 4°C for 15 minutes, after which plasma was removed with a disposable pipette and stored at -20°C until required for analysis. Recovered plasma from lithium heparin tubes was used, along with a slightly modified commercially available RIA kit (MP Biomedicals, LLC., Solon, OH, USA), to measure plasma cortisol concentration. Following kit instructions for volume of sample (25 μL) yielded results below the normal limit of detection (< 10.0 ng/mL). Therefore, different volumes of plasma sample (25, 50, 100, and 150 μL) were evaluated. Using parallel displacement and recovery (101.59 ± 10%) it was determined that using 50 μL of sample yielded results between minimum and maximum concentration of 5.0 and 500.0 ng/mL, respectively. The intra-assay and inter-assay variations were 2.8 and 11.1%, respectively.
Oxytocin concentration were also determined using a commercially available double-antibody RIA kit (Oxytocin (Human, Rate, Mouse, Bovine) RIA kit, Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA) using plasma recovered from EDTA tubes. The inter-assay variation was 19.3% and the intra-assay variation ranged from 3.1 to 9.6%. The minimum and maximum concentrations of detection were 2.5 and 160.0 pg/mL, respectively. The analysis for plasma cortisol and oxytocin were completed using duplicate samples.