Fig 1: (A) Pancreatic sections were double labeled for insulin (green fluorescence) and 4-HNE (a marker for oxidative stress; red fluorescence). ZDF rats after 9 days of a high-fat diet showed intense cytoplasmic staining for 4-HNE in beta cells (B). This was reduced 2 weeks after a return to regular diets (C). Immunostaining for Nrf-2 (red fluorescence) revealed significant immunoreactivity both in the cytoplasm and the nucleus of beta cells ZDF rats fed high-fat diets for 9 days (F) when compared to ZDF controls (E). In contrast, 2 weeks after returning to regular diets the immunoreactivity for Nrf2 was dramatically reduced (G). Similarly, pancreatic sections stained for HO-1 (red fluorescence) showed increased cytoplasmic and nuclear localization of HO-1 (J) in beta cells (green fluorescence), which after 9 days of HFD was greatly diminished 2 weeks after return to regular diet (K). Specificity of detected immunoreactivities was validated by incubation of tissue sections with control IgGs from each species (lower panels). Morphometric analysis of markers of oxidative stress. To determine the presence of oxidative stress, pancreata from both control ZDF rats fed with a 17% fat diet and HFD-treated ZDF rats fed with a 48% fat diet were fixed overnight with 4% formalin. Fixed tissues were processed for paraffin embedding, and 4-µm sections were prepared and mounted on slides. Sections from both groups of animals were chosen at 25-µm intervals for immunolocalization of 4HNE, Nrf2, and HO-1, as well as insulin to identify ß cells and E-cadherin to identify the pancreatic epithelium. Fifty sections per animal group were analyzed for morphometric measurements. Primary antibodies used for these experiments included anti-4HNE (Abcam, ab46545), anti-Nrf2 (Abcam, ab31163), and anti–HO-1 (Enzo Life Sciences), all used at 1:100 dilution and incubated overnight at 4 °C. Following reaction with fluorophore-conjugated secondary antibodies (Jackson ImmunoResearch) slides were mounted with DAPI containing mounting medium (Vector Laboratories) and viewed at a Nikon i90 microscope for image acquisition and analysis using NIS-Elements AR 3.2 (Nikon). *** p < 0.001, **** p < 0.0001. Modified from Ref. [23].