Immunolocalization of Hepatic TNF-α

Immunolocalization for hepatic tumor necrosis factor-α (TNF-α) was applied on sections of 5-μm thickness and stained with the streptavidin-biotin-peroxidase staining technique [20 , 42 (link), 43 (link)] using an antisera containing 1^ry antibodies for rat anti-TNF-α (Santa Cruz, CA, USA), biotinylated 2^ry antibodies and streptavidin horseradish peroxidase (Dako-K0690; Dako Universal LSAB Kit), and diaminobenzidine tetrahydrochloride (DAB) substrate kit (Sigma-D5905; Sigma–Aldrich Company Ltd., Gillingham, UK) for 10 min for immunolabelling. All prepared sections were treated under the same conditions with the same antibody concentrations simultaneously, so the immunostaining among the different groups was comparable.