Nrf2 Knockout Mouse Brain Analysis

All animal care was performed in compliance with The Oklahoma Medical Research Foundation Institutional Animal Care and Use Committee-approved protocol. Nrf2^−/− mice (10–25 months) were backcrossed 10 generations into a C57BL6 background and have been previously described [26 (link), 31 (link)]. C57BL/6 mice were used in the control (1–4 months) versus aged (10–25 months) analysis. Mice were euthanized by CO₂ asphyxiation and decapitated to remove brains. Tissue was fixed in 10 % neutral buffered formalin at 4 °C overnight then processed and paraffinized using an overnight protocol with ethanol, xylene, and paraffin equilibration in a Shandon Excelsior tissue processor (Thermo Scientific). Brains were cut into 7 μm-thick sagittal sections using a Leica Instruments Microtome (Model 2045 Multicut), mounted on charged glass slides, and warmed on a Lab-Line Instruments slide warmer (Model 26020) overnight. A mouse brain atlas [20 ] was used throughout the sectioning process to ensure correct orientation using white matter, cerebellum, and various structural markers as landmarks. These same landmarks were observed to ensure studies compared the same sagittal plane within each brain, although we observed very similar UbcM2 expression levels in various planes of the same substructures within a brain (data not shown).