Quantification of Testicular TNF-α

Testicular levels of TNF-α was estimated as described in previous studies using rat TNF-α ELISA kit (USCN Life Science, Houston, TX, USA) [17 (link), 26 (link)] based on a sandwich enzyme immunoassay. The manufacturer’s instructions were followed. Standards or samples were added to the microtiter plate wells with a biotin-conjugated antibody specific to TNF-α. Afterwards, avidin conjugated to HRP was added and incubated. After the addition of TMB substrate solution, wells that contained TNF-α, biotin-conjugated antibody and enzyme-conjugated avidin would display a change in color. The enzyme-substrate reaction was ended by the addition of sulfuric acid and the color change was determined at 450 nm.