Anti nrf2 sc 722

Protein lysates (20 μg protein) of kidney tissue or cells were separated by SDS-PAGE and blotted on polyvinylidene difluoride membranes (Millipore). The membranes were incubated overnight at 4 °C with the respective primary antibodies and secondary antibodies accordingly. Staining was then developed using the ECL Plus detection system (Amersham Biosciences, Little Chalfont, UK). The antibody against villin was from Chemicom (MAB1639a, Toronto, Canada), anti-tubulin (T6074) was from Sigma-Aldrich (Buchs, Switzerland), anti-Grp78 (ab21685) was from Abcam, anti-ICAM (sc-1511), anti-FXR (sc-13063) and anti-Nrf2 (sc-722) were from Santa Cruz and anti-SDHA (#11998) and anti- cleaved caspase 3 (#9661) were from CellSignaling (Danvers, MA, USA).

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.