Ria kit

After administration of Beuthanasia, N/OFQ levels in CSF and serum were determined by RIA kit (Phoenix Pharmaceuticals, CA) according to the manufacturer’s protocol, and data are presented as N/OFQ-IR. The sensitivity of the assay was < 10 pg/mL; non-specific binding was 2.9%. There was no cross-reactivity with Dynorphin A (1–17), enkephalin, or β-endorphin. Corticosterone levels in serum also were determined by RIA kit (MP Biomedicals, NY) according to the manufacturer’s manual. The sensitivity of the assay was 25 ng/mL and non-specific binding was 2.6%. Total amount of corticosterone was calculated and expressed as ng/mL. Samples and standards were assayed in duplicate. RIA curves and data were analyzed using GraphPad Prism 8.2 software.

Plasma CORT concentrations were measured using a commercial radioimmunoassay (RIA) kit from MP Biomedicals Inc. (Solon, OH, USA). Plasma insulin, leptin contents were determined with specific mouse enzyme-linked immunosorbent assay (ELISA) kits (Millipore, Billerica, MA, USA). For plasma fibroblast growth factor 21 (FGF21), ELISA kit (BioVendor, Candler, NC, USA) was applied to measure the concentrations. All measurements were performed according to respective manufacturer's instructions.
The concentrations of triglyceride, NEFA and malondialdehyde (MDA) were measured using commercial assays (Jiancheng Institute of Bioengineering, Nanjing, Jiangsu, China) as described previously [27] (link). The activities of superoxide dismutase (EC Number: 1.15.1.1), gluthathione peroxidase (EC Number: 1.11.1.9) and catalase (EC Number: 1.11.1.6) were measured using specific commercial kits (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China) according to the provided instructions, respectively.

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.

To determine basal plasma corticosterone hormone levels, blood sampling was performed in the early morning (08:30–09:30 h) and afternoon (04:30–05:30 h) by collecting blood from the tail vein. Samples were collected in 1.5 ml EDTA-coated microcentrifuge tubes (Kabe Labortechnik, Germany). All blood samples were kept on ice and later centrifuged at 8000 rpm at 4°C for 15 min. Plasma was transferred to new labeled microcentrifuge tubes and stored at −20°C until further processing. Plasma corticosterone concentrations were measured using a commercially available RIA kit (MP Biomedicals, Eschwege, Germany) according to the manufacture’s manual.