Corticosterone enzyme immunoassay kit

Mice were euthanized by cervical dislocation immediately followed by decapitation. Trunk blood was collected in an EDTA-coated tube. Plasma was separated by centrifugation at 600 g for 15 min at 4 °C and stored at −80 °C. Corticosterone levels were measured in duplicates using the Corticosterone Enzyme Immunoassay Kit (Arbor Assays, Ann Arbor, MI) according to the manufacturer’s instructions.

Quantification of fecal corticosterone was performed as described previously (26 (link)). Feces from mice maintained in individual cages were collected at four time points: baseline, after STZ, acute CRS, and chronic CRS (Figure 1A). Strip litter (Pulsoft, Oriental Yeast Co.; Pulmas μ, Material Research Center Co., Kawasaki, Japan) was used to ensure effective collection of feces, which were stored at −80°C until analysis. Fecal samples were completely dried, weighed, and thoroughly ground. One milliliter aqueous ethanol [ethanol: deionized distilled water (DDW), 8:2 (v/v)] was added to 50 mg fecal powder, boiled (99°C) for 5 min, and vortexed for 10 min. The boiling and vortexing steps were repeated two additional times. After centrifugation (10 min at 2,500 × g), the supernatant was transferred into another tube and stored at −20°C until performing the ELISA assay. We used a Corticosterone Enzyme Immunoassay Kit (Arbor Assays, Ann Arbor, Michigan) according to the manufacturer’s protocol. An aliquot of 2.5 μL supernatant was added to one well of the ELISA plate per animal.

Blood was collected from the tip of the tails, and blood glucose levels were measured with an Antsense Duo Small Electrode Glucose Analyzer (Horiba, Kyoto, Japan). Blood samples were centrifuged (13,500 rpm, 10 min, 4 °C) twice, and the collected plasma samples were stored at −80 °C until the biochemical analyses. Plasma hormone levels were measured using the following assays: insulin, Mouse/Rat Insulin ELISA Kit (Morinaga Institute of Biological Science, Kanagawa, Japan); FGF21, Mouse and Rat FGF21 ELISA Kit (BioVendor Inc., Brno, Czech Republic); GCG, Glucagon ELISA Kit (Mercodia, Uppsala, Sweden); active GLP-1, GLP-1 Active Form Assay Kit (IBL, Gunma, Japan); GIP, GIP (total) ELISA kit (Millipore, Billerica, MA, USA); and corticosterone, Corticosterone Enzyme Immunoassay Kit (Arbor Assays, Ann Arbor, MI, USA). Plasma amino acid concentrations were measured by standard assays at SRL, Tokyo, Japan [21 (link)].

Corticosterone levels in serum samples were measured without any extraction procedure using the Corticosterone Enzyme Immunoassay kit (Arbor Assays, Ann Arbor, MI, United States). Concentrations were calculated using a 4-parameter curve and expressed in ng/ml.