Shandon excelsior

Samples fixed in neutral-buffered formalin were trimmed, followed by a dehydration and clearing step in an automatic tissue processor (Thermo Scientific^TM Shandon Excelsior, Kalamazoo, MI, USA). The samples were then embedded in paraffin wax and sectioned using cryotome. Sectioned samples were then stained with hematoxylin and eosin; and observed under light microscope.

Tumours and mammary glands were fixed in 4% paraformaldehyde (PFA: Scharlau FO) for 24 h at room temperature and washed in 70% ethanol before being embedded in paraffin for automated processing (Shandon Excelsior, Thermo). The samples were sectioned and stained with haematoxylin and eosin to evaluate their pathology under a microscope. Five photos (10× magnification) were taken randomly with a Leica ICC50 HD camera under the control of Leica Application Suite V3.7 software, quantifying the relative ductal area of the mammary glands. Image analysis was performed using ImageJ, selecting the ductal area (epithelial cells forming the duct) while excluding the adipose tissue and duct lumen. The ductal epithelial area was divided by the total field area to calculate the relative percentages, and the mitotic index of the tumours was defined as very high if there were more than eight mitoses at 40× magnification, high if there were between four and eight mitoses, moderate when there were between two and four mitoses, and low if there were less than two mitoses. A pathologist evaluated this parameter in the Pathology Unit of our Centre.

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).

Tumors and mammary glands were fixed in 4% paraformaldehyde (PFA: Scharlau FO) for 24 h at room temperature and washed in 70% ethanol before being embedded in paraffin for automated processing (Shandon Excelsior, Thermo). The samples were sectioned and stained with hematoxylin and eosin to evaluate their pathology under a microscope. Five photos (10x magnification) were taken randomly with a Leica ICC50 HD camera under the control of Leica Application Suite V3.7 software, quantifying the relative ductal area of the mammary glands. Image analysis was performed using ImageJ, selecting the ductal area (epithelial cells forming the duct) while excluding the adipose tissue and duct lumen. The ductal epithelial area was divided by the total field area to calculate the relative percentages, and the mitotic index of the tumors was defined as very high if there were more than eight mitoses at 40x magnification, high if there were between four and eight mitoses, moderate when there were between two and four mitoses, and low if there were less than two mitoses. A pathologist evaluated this parameter in the Pathology Unit of our Center.

Mammary glands were fixed in 4% paraformaldehyde (PFA) and processed for paraffin embedding (Shandon Excelsior, Thermo). Tissue sections were stained with hematoxylin and eosin for morphological examination. Quantitative analysis of adipocyte and ductal areas was conducted on five randomly selected images at 10x magnification per sample using a Leica ICC50 HD camera and Leica Application Suite V3.7 software. ImageJ was employed to determine the relative ductal epithelial area as a percentage of the total field area.

Hearts were fixed in 4% paraformaldehyde (Scharlau FO) for 24 h and then processed in an automatic system (Shandon Excelsior, Thermo Fisher Scientific, Waltham, MA, USA). The subsequent samples were sectioned, embedded in paraffin, and stained with hematoxylin-eosin with a standard protocol or the Masson-Goldner Trichrome kit (Bio-Optics, Daejeon, Republic of Korea) to evaluate the cardiac fibrosis and cardiomyocyte area. We robotically quantified heart fibrosis and the average area of myocardial fibers as pathophenotypes of CDA using the Ariol slide scanner to avoid intra- and inter-observer deviations. Histopathological damage was measured in the subendocardium and subepicardium from five randomly chosen regions of each sample. Regarding protocols for quantifying intermediate molecular phenotypes, see the Supplementary Methods section.