Progres c5 camera

Dissected mammary tumors, mammary glands and lungs were fixed in 10% neutral buffered formalin and embedded in paraffin wax. Sections (6μm) were deparaffinized in xylene (2×5min) and rehydrated in descending concentrations of ethanol (2×100%, 95%, 70%; 5min) before being placed in ddH₂O (2×3min). Tissues were then stained in 1% Harris's Hematoxylin for 1 min before being washed in tap water and differentiated in acid ethanol. Slides were dipped in 70% ethanol (30sec) and put into alcoholic Eosin (2min) and finally placed back into 70% ethanol (2×30sec). Slides were then dehydrated and mounted using Cytoseal. Images were captured using a Brightfield microscope equipped with a ProgRes C5 camera (Jenoptik) and ProgRes Mac CapturePro 2.7.6 imaging software. Multiple rounds of sectioning and staining were used to evaluate lung histology for evidence of lung metastases. Clusters of nuclei in intimate proximity with bronchioles, typical of bronchus associated lymphatic tissue, were not included in the quantification of lung tumors. Lung tumour area was evaluated by measuring the length and width of lung tumors which were used to determine the area of an ellipse using ImageJ and the area of multiple lung tumors per mouse was averaged (National Institutes of Health, Bethesda, MD).

The subcellular localization assay was performed as previously described [18 (link)]. Cells (2 × 10⁵ cells/well) were seeded onto 12 mm coverslips in 12-well plates and subsequently treated with either the vehicle control (DMSO), HmARE, or sulforaphane (SFN). After 6 h, the cells were fixed using 4% paraformaldehyde (Sigma Aldrich, St. Louis, MO, USA) in PBS (pH 7.4) for 15 min. Following PBS washing, permeabilization was achieved by immersing the cells in a 0.3% Triton X-100 solution in PBS for 10 min, followed by blocking with normal goat serum (Vector Lab., Burlingame, CA, USA). Subsequently, the cells were incubated with anti-Nrf2 rabbit IgG (Cell Signaling, Beverly, MA, USA), followed by incubation with anti-rabbit IgG conjugated with FITC (Life Technology, Eugene, OR, USA). The cells were mounted using a mounting medium containing DAPI (Abcam, Cambridge, MA, USA) for nuclear counterstaining. Fluorescent images were captured using a Zeiss Axioskop 50 fluorescence microscope (Carl Zeiss, Jena, Germany) equipped with a ProgRes C5 camera (Jenoptik, Jena, Germany).