Quantification of ecdysteroids in whole larvae was performed as described by [61 (link),113 (link)] with the following modifications. Briefly, animals were homogenized in 0.25 ml 75% methanol, and then the supernatants were collected following centrifugation at 14,000 g for 15 min. The pellets were re-extracted in 0.1 ml methanol. The supernatants were combined, evaporated using a SpeedVac, and then re-dissolved in 0.5 ml ELISA buffer (Cayman Chemical). Ecdysteroids were measured using a commercial ELISA kit (Cayman Chemical) that detects 20E equivalents. Standard curves were generated using 20E (Cayman Chemical), and absorbance was measured at 405 nm on a microplate photometer (Thermo Scientific).
Elisa buffer
Manufactured by Cayman Chemical
Sourced in United States
ELISA buffer is a laboratory reagent used in enzyme-linked immunosorbent assays (ELISA). It is designed to maintain the optimal pH and ionic conditions for the specific binding and detection reactions that occur during ELISA experiments.
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5 protocols using elisa buffer
1
Ecdysteroid Quantification in Insect Larvae
2
Quantifying Anti-inflammatory Compounds
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Injectable indomethacin (INDOCIN®) was purchased from Iroko Pharmaceuticals LCC (Philadelphia, PA, USA) and injectable acetaminophen (Mol) was purchased from Gufic Biosciences. Clean tubes, carbonate buffer, phosphate buffer, mouse monoclonal anti-rabbit IgG, wash buffer, Ellman’s reagent, and ELISA buffer were purchased from Cayman Chemical, USA. Microwells were purchased from Sigma-Aldrich (St. Louis, MO, USA).
3
Crab's Reproductive Hormone Dynamics
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In every month of the year 2012, EDs from 3 crabs (300 ± 20 g) were tested for PG, testosterone, and estradiol levels using ELISA kits (Cayman Chemical Company, Ann Arbor, MI, USA). Tissues (~50 mg) were ground in liquid nitrogen and mixed with 1 mL of ELISA buffer (Cayman Chemical Company, Ann Arbor, MI, USA); supernatants were subjected to ELISA following the manufacturer’s instructions. Analyses were carried out in duplicate. For evaluation of PG level in pre- and post-mating females, spermathecae and ovaries from un-mated females, female crabs at the day after mating, post-mating stage I, II, III, pre-ovulation, and post-ovulation stage, were collected (n = 6). The samples (~30 mg) were analyzed as described above.
4
Testicular Fluid Extraction for Testosterone
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Using a 27-gauge needle, the tunica of the testes was punctured 3 to 4 times opposite the rete testis and centrifuged in a 1-mL pipet tip at 500 × g for 10 minutes. Interstitial fluid collected in the bottom of the tube was diluted in ELISA buffer (Cayman Chemicals) for testosterone measurement by ELISA.
5
Corticosterone Measurement in Blood and Saliva
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For both blood and saliva, each sample was measured in duplicate by using a commercially available EIA kit (mouse anti-rabbit IgG, Corticosterone ELISA kit, number 501320, Cayman Chemicals) previously used and validated for different bird species [39, (link)40] (link). The assay was conducted following the manufacturer recommendations and samples were reconstituted and diluted (1:50 for blood samples, and 1:25 for saliva samples) with Cayman Chemical ELISA buffer to ensure that concentrations fell within the linear portion of the standard curves. Samples were run across 2 EIA plates with inter-assay coefficients of variation of 6.32% (high concentration) and 8.96% (low concentration), and intra-assay coefficients of variation of 7.8% (range = 0.2 -12.9%). For each sample, mean values of salivary and plasma corticosterone concentrations were used for statistical analysis.
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