Ab46545

Five-micron sections of formalin–fixed, paraffin-embedded lung tissue were stained with hematoxylin and eosin by standard procedures at the University of Colorado Histology Core to assess vessel wall thickness. Immunohistochemistry analysis for α-smooth muscle actin, 4-hydroxynonenal, interlukin-6 (IL-6) was performed per the manufacturer’s instructions. Antigen retrieval was performed on serial lung sections and then incubated with antibodies against smooth muscle actin (1:500 abcam, ab5694, Cambridge, MA), 4HNE (1:500 abcam, ab46545 Cambridge, MA) and IL-6 (1:50, Santa Cruz, sc-1265). Lung sections were incubated with a fluorescent secondary antibody (either Alexa Fluor 488 goat anti-rabbit IgG (1:300 or 1:800; A11008) or Alexa Fluor 555 (1:500; A21422) Invitrogen, Carlsbad, CA). Primary antibodies were incubated at 4°C overnight, and Secondary antibody was incubated for 1 hour at room temperature. Slides were then mounted with Vectashield mounting medium for fluorescence with dapi (H-1200) from Vector and coversliped. Pulmonary vessels (outside diameter ~100 μm) were assessed for the presence of smooth muscle actin, 4HNE and IL-6 protein expression on a Nikon Eclipse Ti-E inverted epi-fluorescent microscope (Nikon Instruments, Tokyo Japan).

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).