Mab424r

Kidney sections (3 µm) (RM 2164, Leica, Wetzlar, Germany) were mounted on glass slides, heated for 1 h at 60 °C, and deparaffinized with Roti-Histol (Roth, Karlsruhe, Germany) and ethanol. Antigen retrieval was performed with citrate buffer (DAKO Retrieval Solution, pH 6.0, Agilent Technologies, Santa Clara, CA, USA) at 95 °C for 30 min. Afterwards the slides were blocked and incubated with the corresponding primary antibodies at 4 °C overnight. The specific antibodies and dilutions were as follows: anti-γ-H2AX (#9718, 1:200, Cell Signaling, Herts, UK), anti-Nrf2 (sc-722, 1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), anti-pNrf2 (phospho S40, ab76026, 1:1000, abcam, Cambridge, UK), anti-NQO1 (ab34173, 1:500, abcam, Cambridge, UK), and anti-PCNA (MAB424R, 1:1000, Merck, Darmstadt, Germany). Subsequently, the sections were incubated with the biotinylated secondary antibodies goat anti-rabbit (ab6720, 1:200, abcam, Cambridge, UK) or goat anti-mouse (ab6788, 1:200, abcam, Cambridge, UK) for 45 min at room temperature. Antibody binding was visualized as described before [21 (link),28 (link)]. Sections were counterstained with hematoxylin. Pictures were taken at 200-fold magnification. The ratio of positive to negative nuclei or area was assessed via Image J (http://imagej.nih.gov/ij/index.html) within 10–15 visual fields of the cortex and 3–5 visual fields of the medulla.

Zebrafish for histological analysis were euthanized with tricaine, fixed in 4% paraformaldehyde at 4°C overnight, and decalcified with 0.25 M EDTA (pH 8.0). Paraffin sectioning followed by hematoxylin and eosin (H&E) staining or immunohistochemistry was performed at the Dana-Farber/Harvard Cancer Center Research Pathology Core. Primary antibody (PCNA, EMD Millipore, MAB424R, 1:100) binding was detected with a diaminobenzidine-peroxidase visualization system (EnVision+, Dako). Mayer’s hematoxylin was used for counterstaining. Slides were imaged using an Echo Revolve4 inverted microscopy system.