Lod 2.5 pg mlt mouse rat kit

Shortly before the sacrifice, blood was collected by intracardiac puncture with stratification across groups to reduce the potential impact of circadian rhythm and pulsatility. Blood samples were left to coagulate at room temperature for 1 h, centrifuged for 15 min at 2000 rpm twice in a cooled bench-top centrifuge (Microlite Microfuge, Thermo Electron Corporation) and stored at -80 °C until use. Serum levels of all hormones were measured in the same analytical section by the following commercial ELISA kits of the same lot(s):

E2 Rat kit (RTC009R—BioVendor Brno, Czech Republic), LOD 2.5 pg/ml

T Mouse/Rat kit (RTC001R—BioVendor Brno, Czech Republic), LOD 2.5 pg/ml

TSH Rat Kit (ELK2283—ELK Biotechnology, China), LOD 0.071 ng/mL

T4 Rat Kit (ELK8716—ELK Biotechnology, China), LOD 1.42 ng/mL

Each kit provided a standard solution of the hormone and serial dilutions were prepared to derive a standard curve and define the range of linearity of each test. For all the analyses, the manufacturer’s instructions were followed. Each sample was assessed in duplicate and the absorbance was read at 450 nm on a VICTOR3 Multilabel reader (Perkin Elmer, USA). The unknown hormone concentrations in samples were derived using the standard curve of each hormone and the software GraphPad Prism 5.0 (GraphPad Software Inc.).

Blood was always sampled from 9 to 10 am, with stratification across treatment groups, in order to reduce the potential impact of circadian rhythm and pulsatility. Blood samples were left to coagulate at room temperature for 1h, centrifuged twice for 15 min at 2000 rpm in a cooled bench-top centrifuge (Microlite Microfuge, Rodano (MI), Italy) and stored at −80 °C until use. Serum levels of all hormones were measured in the same analytical section by the following commercial ELISA kits of the same lot(s):-

E2 Rat kit (RTC009R—BioVendor Brno, Czech Republic), LOD 2.5 pg/mL

-

T Mouse/Rat kit (RTC001R—BioVendor Brno, Czech Republic), LOD 2.5 pg/mL;

-

LH Rat Kit (ELK2367—ELK Biotechnology, Whuan, China), LOD 37.59 pg/mL

Each kit provided a standard solution of the hormone, and serial dilutions were prepared to derive a standard curve and define the range of linearity of each test. For all the analyses, the manufacturers’ instructions were followed. Each sample was assessed in duplicate, and absorbance was read at 450 nm on a VICTOR3 Multilabel reader (Perkin Elmer, Milan, Italy). The unknown hormone concentrations in samples were derived using the standard curve of each hormone using the software GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA).