Corticosterone enzyme immunoassay kit

Blood was collected from the tip of the tail and blood glucose levels were measured with an Antsense Duo Small Electrode Glucose Analyzer (Horiba, Kyoto, Japan). Blood samples were centrifuged (2000× g, 20 min, 4 °C) and the collected plasma samples were stored at −80 °C until analysis. Plasma hormone levels were measured using the following assays: insulin, Mouse/Rat Insulin ELISA Kit (Morinaga Institute of Biological Science, Kanagawa, Japan, Cat# M1108); FGF21 (Fibroblast growth factor 21), Mouse and Rat FGF21 ELISA Kit (BioVendor Inc., Brno, Czech Republic, Cat# RD291108200R); corticosterone, Corticosterone Enzyme Immunoassay Kit (ARBOR ASSAYS, Ann Arbor, MI, USA, Cat# K014-H1); FFA, NEFA C test (FUJIFILM Wako Pure Chemical Co., Osaka, Japan, Cat# 279-75401); triglyceride (Triglyceride E test, FUJIFILM Wako Pure Chemical Co., Osaka, Japan, Cat# 432-40201); and total cholesterol (LabAssay Cholesterol, Cholesterol Oxidase·DAOS method) (FUJIFILM Wako Pure Chemical Co., Osaka, Japan, Cat# LABCHO-M1).

All feces obtained within the 2 h sampling time was placed in a Petri dish and dried at 60 °C for 2 days. Then, the samples were stored frozen until the extraction operation was performed. To extract corticosterone (CORT) in feces, dried feces were crushed and weighed at portions of 40 mg each in a 2.0 mL tube, and then were dispensed 1 mL of methanol. After that, it was stirred at room temperature for 24 h using a rotator. The stirred sample was centrifuged at 10,000× g at room temperature for 10 min. Then, the supernatant was transferred to a test tube (13 × 100 mm) and dried with nitrogen gas for about 40 min while heating at 38 °C in a heat block (Dry Thermo Unit1C, TAITEC, Koshigaya, Japan). PBS (0.3 mL) was added, and the mixture was shaken at room temperature for 1 h using the shaker. After shaking, it was transferred to a microtube and stored at −30 °C until measurement [30 (link),31 (link)]. The concentration of corticosterone in the extracted feces was measured using an ELISA measurement kit (Corticosterone Enzyme Immunoassay Kit, ARBOR ASSAYS, Ann Arbor, MI, USA). Visible light density was measured at a wavelength of 450 nm using a microplate reader (I Mark microplate reader, Bio-Radola Volunteers Co., Ltd., Tokyo, Japan).