Fig 1: Formation of HNE-adducts on vimentin in murine skin fibroblasts. Cultured murine fibroblasts were exposed to HNE (30 µM, 4 h in HBSS medium). At the end, cells were fixed in paraformaldehyde (4% in phosphate buffered saline, PBS, for 10 min), and then used for confocal imaging studies, in the previously reported conditions [67]. After blocking with PBS containing 5% bovine serum albumin for 45 min, cells were incubated with the primary anti-HNE-Michael adduct antibody (#MA5-27570, Invitrogen), or the anti-vimentin antibody (#92547, Abcam), followed by Alexa Fluor-488 or Alexa Fluor-546 conjugated antibodies. Representative confocal imaging pictures show the presence of HNE-adducts (green, right panel) on vimentin (red, middle panel), and the merge (yellow, left panel), in fibroblasts incubated with HNE (lower pictures) vs. control without HNE (upper pictures). Scale bar = 5 µm. Original 63× confocal images are shown inset.