Immunohistochemical Analysis of Liver Markers

Briefly, liver sections (4 µm thick) were subjected to antigen retrieval and block of endogenous peroxidase activity and avidin-biotin-peroxidase method according to previous study [40 (link)]. The slides were incubated with normal serum from species producing the secondary antibody and subsequently with primary polyclonal antibodies against 4HNE (1:400, Abcam, #ab46545, Cambridge, UK), SIRT1 (1:150, Santa Cruz Biotechnology, #sc15404, Dallas, TX, USA), GRP78 (1:300, Abcam, #ab21685), SREBP1 (1:100, Santa Cruz Biotechnology, #sc8984), IL6 (1:100, Santa Cruz Biotechnology, #sc1265), p62/SQSTM1 (1:50, MBL International, Woburn, MA, USA) or monoclonal antibodies against F4/80 (1:50, Bio Rad, #MCA497GA, Segrate, Italy), Mitofusin 2 (1:200, Abnova, #H00009927, Taipei, Taiwan). All experiments were performed in triplicate. The staining intensity was expressed as arbitrary units (AU) or percentage of positive nuclei, in 20 randomly chosen microscopic fields, using an image analyzer (Image Pro Premier 9.1, Media Cybernetics, Rockville, MD, USA).