Eia kit

Plasma was stored at −20°C and then assessed for Blood Ethanol Concentrations (BECs) using an Analox AM-1 alcohol analyzer, and for corticosterone (ADI-900-097) and progesterone (ADI-901-011) using EIA kits (Enzo Life Sciences). CORT intra-assay coefficient of variability (CV) was 1.01, and inter-assay CV was 8.93. PROG intra-assay CV was 1.20, and inter-assay CV for was 1.61. Progesterone was measured in Experiment 1 as a secondary index of neuroendocrine responses to ethanol challenge (Hueston and Deak, 2014 (link)), and its potent immunomodulatory effects (Hueston and Deak, 2020 (link)).

Plasma concentrations of sP-Selectin/CD62-P, Serpin E1/PAI-1, and serum concentrations of sE-Selectin/CD62E, sICAM-1/CD54, sVCAM-1/CD106, and VEGF were measured using commercial ELISA kits (R&D Systems® Europe, Ltd., UK) according to the manufacturer's instructions.
Plasma concentrations of prostanoids (6-keto-PGF-1 alpha as a marker of prostacyclin synthesis and TXB₂ reflecting thromboxane formation) were measured using commercial EIA kits (Enzo Life Sciences GmbH, Germany) according to the manufacturer's instructions.
Carotid-femoral, carotid-radial, and carotid-dorsal pedis pulse wave velocities (PWV) were assessed using automatic Complior SP device (s/n 200901-000612, Artech Medical, France) and dedicated software (ver. 1.3.0). PWV (m/s) was calculated by dividing the distance between arteries by the time shift (transit time). After blood pressure measuring, four piezoelectric Complior transducers were placed on skin over the carotid, radial, femoral, and dorsalis pedis artery. After acceptable signal strength and quality had been obtained, pulse wave data was acquired and computer calculation was performed.

Tail blood samples were taken immediately following the first two sucrose preference tests, which corresponded to proximal (4 days) and post-washout (14 days) time points following the cessation of CORT exposure. At the end of the experiment, rats were euthanized via rapid decapitation, and blood samples were collected in heparinized 1.5 ml centrifuge tubes. Blood samples were collected during the latter half of the light cycle. Plasma from each time point was analyzed for CORT using commercially available EIA kits (Enzo Life Sciences, Farmingdale, NY, United States) according to the manufacturer’s instructions.

We used plasma samples from the spring and summer seasons, when birds were either coming into, or in, reproductive condition, for the assessment of reproductive hormone concentrations. We first validated enzyme immunoassay (EIA) kits from Enzo Life Sciences (Farmingdale, NY, USA) for testosterone (ADI-900-065) in samples from male birds and for estradiol (17β-estradiol high sensitivity; ADI-900-174) in samples from female birds, for each of the 11 species. We used pooled plasma samples in 10, 20, 30 and 40 µl volumes, using appropriate amounts of assay buffer in each case to ensure that the total volume in each well was the same for each sample and running each in duplicate to optimize the assays for each species. Beyond the sample volume adjustments, the remainder of each assay was completed following the manufacturer's recommendations. Final hormone concentrations for the samples were determined by comparing the values from the average of duplicate samples with a nine-point standard curve generated by the manufacturer's provided standards. Intra-assay coefficients of variation (CVs) were calculated from six standard samples included in each plate, yielding average CVs of 7.21% among the plates and intra-assay CVs of 11.35% for testosterone and 8.89% for estradiol.

Corticosterone levels were measured in H, N-H, RCF and N-RCF male mice, at different time intervals from novelty exposure. Apart from the postnatal manipulation, these animals have never been exposed to other experimental procedures. Novelty consisted in exposing the animals to a novel environment: each mouse was removed from its home cage and placed in the center of an open circular arena (60 cm diameter) for 20 min. Trunk blood samples were collected at different time intervals after the novelty test. One group of animals for each treatment was not manipulated at all and blood collected represented the group baseline (Time 0′). Immediately at the end of the novelty exposure, 50% of mice were sacrificed to measure the stress response to the open arena (Time 20′), while the other 50% was reintroduced in their home cages and blood was collected after 40 min (Time 60′). After blood centrifugation (20 min, 4°C, 4000 rpm), serum samples were stored at −25°C until assay were conducted. Corticosterone levels were measured using commercially available EIA kits (Enzo Life Science, sensitivity 27.0 pg/mL). All corticosterone measures were carried out in duplicate. The mean serum corticosterone levels of mice were compared by a two-way ANOVA, the factors being (1) manipulation (4 levels: H, N-H, RCF and N-RCF); and (2) time intervals (3 levels: time 0, 20′ and 60′).