Igf 1 standards

IGF-1 standards (Abcam, Cambridge, UK) and sera samples were diluted in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, 3 mM NaH3, pH9.6) and allowed to bind to Costar (3590) high-binding EIA/RIA plates overnight. Subsequently, plates were washed 3 times in PBS-0.1% Tween-20 (PBST), blocked with 3% milk in PBST (PTM) for 1 h and incubated in primary antibody, rabbit anti-IGF-1 in PTM 1:2000 (Abcam, Cambridge, UK) for 2 h at 37 °C. Next, plates were washed 3 times in PBST and incubated in secondary antibody goat antirabbit IgG1 conjugated to HRP in PTM 1:4000 (Southern Biotechnology, Birmingham, AL) at 37 °C for 1 h. Plates were then washed in PBST, incubated in TMB peroxidase substrate (Rockland) for 10 min at room temperature, followed by stop solution (2 N H2SO4). Absorbance was read at 450 nm.