Immunostaining of Liver Tissue

Deparaffinized liver sections were used for immunostaining of TNF-α and Caspase-3 as described previously [45 (link)]. Caspase-3 immunostaining was done using rabbit polyclonal anti-cleaved Caspase-3 antibody diluted 1:100 (BioCare Medical, Cat. CP229C, Concord, CA, USA). TNF-α immunostaining was done using a polyclonal rabbit anti-TNFα antibody diluted 1: 400 (GeneTex, Cat# GTX110520). Twenty sections were examined in each group. To determine the Caspase-3 and TNF-α immunoreactivity, the DAB-stained cytoplasmic option in the IHC profiler Image-J plugin was used as described previously [46 (link)] in the captured photomicrographs. Finally, the area% of Caspase-3 and TNF-α expressions was measured using Image J software (freely available public domain image processing software) as described previously [47 (link)].