Dako retrieval solution

Kidney sections (3 µm) (RM 2164, Leica, Wetzlar, Germany) were mounted on glass slides, heated at 60 °C for 1 h 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. Slides were then blocked and incubated overnight at 4 °C with the appropriate primary antibodies. 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) and anti-pNrf2 (S40, ab76026, 1:1000, abcam, Cambridge, UK). Sections were next incubated with the biotinylated secondary goat anti-rabbit antibody (ab6720, 1:200, abcam, Cambridge, UK) for 45 min at room temperature. Antibody binding was visualized as previously described [11 (link),29 (link)]. Sections were counterstained with hematoxylin. Images were acquired at 200-fold magnification. The ratio of positive to negative nuclei or areas was scored via ImageJ [30 (link)] within 10 visual fields of the cortex and 3–5 visual fields of the medulla.

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.

Sample aliquots were incubated in 4% paraformaldehyde in PBS for 24 h and embedded in paraffin. 6 µm slides were used and treated with DAKO retrieval solution (pH = 9, Agilent, Santa Clara, CA, USA) for 30 min by applying hot steam. Adipocytes were stained with anti-perilipin-1 (goat, 1:200, Abcam, Cambridge, UK, #ab61682) and macrophages with anti-Iba1 (rabbit, 1:500, WAKO, #019-19741) at 4 °C overnight. Supervised automated analysis was performed using CellSens software (OLYMPUS Life Science, Shinjuku, Japan) to assess adipocyte count and diameter as well as counts of macrophages (n = 60).