Corticosterone eia kit

Corticosterone concentration in the serum was measured by using an enzyme immunoassay kit (Corticosterone EIA Kit, Cayman Chemical Company) according to the manufacturer’s instruction. Serum was directly diluted in the EIA buffer at 50 times dilution. Crossreactivity of the first antibody was 100% to corticosterone, 0.31% to progesterone, 0.06% to aldosterone, 0.03% to testosterone, 0.02% to pregnenolone, 0.01% to 5α-dihydrotestosterone and less than 0.01% to androstenedione.

The DEXamethasone (DEX)/corticotropin releasing hormone (CRH) test was conducted according to the method described by Hatzinger et al. [21] (link). Surgery was performed under sodium pentobarbital (40 mg/kg, i.p.) anesthesia using aseptic procedures. Rats were chronically catheterized in the jugular vein for subsequent blood sampling six days before the experiment. The catheter was exteriorized at the neck of the animal and filled with sterile saline containing gentamicin (30,000 IU/rat); 0.2 ml was infused into the animal. Rats were weighed at 0700 on the day of the experiment. The jugular venous catheter was connected via free-moving devices (Eicom, Kyoto, Japan) to a plastic syringe filled with sterile heparinized saline (50 IU/ml) at 0800. DEX (Sigma, St. Louis, MO) (30 µg/kg, 0.5 ml/kg) was administered intravenously (i.v.) at 1200. Numerous 0.2-ml blood samples were collected at 1800, 1830, 1900 and 1930 to monitor the effects of the DEX treatment on the basal plasma concentrations of corticosterone during the diurnal acrophase. At 1931, human-CRH (kindly donated from Yoshitomi Pharma, Osaka, Japan) (50 ng/kg, 0.5 ml/kg i.v.) was injected. To assess the CRH-stimulated corticosterone secretion, further blood samples were taken at 1940, 2000, 2020, and 2040. Corticosterone was measured by an enzyme immunoassay using a corticosterone EIA kit (Cayman, Ann Arbor, MI).

To measure corticosterone (CORT), one of the primary stress hormone in rats, blood samples were collected by tail nick during the 20^th minute of restraint or control procedures. This sampling occurred between 9–11 AM, when levels are near their basal diurnal state. Blood (approximately 300 µL) was placed in a tube containing heparin, and immediately placed on ice, then centrifuged at 3200 rpm for 5 min at 4°C. Clear plasma was pipetted out, put into tubes and frozen at −20°C until analysis. Samples were analyzed by enzyme immunoassay for corticosterone (Corticosterone EIA kit, Cayman Chemicals), following the manufacturer's suggested protocol. Absorbance was measured and quantified against known concentrations of corticosterone (405 nm; Novostar multiwell plate reader, BMG LabTechnologies, Ortenberg, Germany). All samples were run in duplicate. Corticosterone concentration in samples were interpolated from an exponential fit to percent bound ÷ maximal bound (%B/Bmax) curve from known concentrations and expressed as pg/mL. For calculation, absorbance measures of blank controls and non-specific binding controls were subtracted out from all values.

Animals were euthanized with an overdose of pentobarbital on the next day of the behavioral test. Blood was collected into EDTA (ethylenediaminetetraacetic acid) tubes via cardiac puncture under deep anesthesia. The plasma was collected by centrifugation of the blood at 1,500 rpm for 5 min at 4°C and kept at −80°C until testing. The concentration of CORT, DA, and the GDNF in plasma was detected using a Corticosterone EIA Kit (Cayman, Germany), Rat dopamine, DA ELISA kit (R&D, USA) and a Rat GDNF ELISA kit (Sigma, USA) following the manufacturers manual instructions, respectively.

The level of androgens, in serum, was referred to as T+DHT considering that the anti-testosterone serum №250 showed 100% cross-reactivity with DHT (for references, please see [20 (link),22 (link)]). Serum androgen levels were measured via radioimmunoassay. All samples were measured in duplicate in one assay (sensitivity: 6 pg per tube; intra-assay coefficient of variation 5–8%). Serum corticosterone (CORT) levels in all samples were measured in duplicate in one assay using the corticosterone EIA Kit (Cayman Chemical, Michigan, MI, USA) with 30 pg/mL as the lowest standard significantly different from blank.

Mice were sacrificed with an overdose of sodium pentobarbital (100 mg/kg) at 4 h (16:00) and 48 h (12:00). Heparinized blood was collected by cardiac puncture, centrifuged, and the plasma was removed and stored at −80 °C until analysis. TNF-α and corticosterone levels were determined using Mouse TNF-alpha DuoSet ELISA Kit (R&D systems Inc, Minneapolis, Canada) and Corticosterone EIA Kit (Cayman Chemical Company, Ann Arbor, USA), respectively, according to the manufacturers’ instructions. For the ELISA of corticosterone, plasma samples were first 200× diluted in assay buffer. The detection limit of the TNF-α ELISA assay was 31 pg/ml, and that of the corticosterone ELISA assay was 30 pg/ml (i.e., 6 ng/ml in the undiluted sample).

Androgen concentration was determined by RIA in serum and cell medium samples (17 (link)). Antitestosterone serum number 250 used in this study showed 100% cross-reactivity with testosterone and dihydrotestosterone but recognized also other androgens. Samples were measured in duplicate (sensitivity: 6 pg/tube; intraassay coefficient of variation: 5–8%; interassay coefficient of variation: 7.5%). For serum corticosterone levels (21 (link)), all samples were measured in duplicate in one assay by the corticosterone EIA Kit (Cayman, Ann Arbor, MI, USA) with 30 pg/ml as the lowest standard significantly different from the blank.