Ab236469

After deparaffinization, mouse and human paraffin sections were placed in citrate-buffered solution (pH 6.0) and boiled for 5 min to retrieve antigens. Endogenous peroxidase was quenched with 3.0% hydrogen peroxide in methanol for 20 min. Samples were blocked with 3% BSA in PBS for 30 min at room temperature and incubated with primary antibodies (Supplementary Table 2). Sections were labeled with a goat anti-rabbit HRP-conjugated secondary antibody (ab236469, Abcam). Diaminobenzidine chromogenic substrate (K3468, Agilent Technologies, Inc., Santa Clara, CA) was used for the color reaction, followed by counterstaining with hematoxylin. All sections were observed using an Eclipse E600 microscope (Nikon, Tokyo, Japan) and BZ-X700/BZ-X710 microscope (Keyence Corporation, Osaka, Japan). γH2AX-positive cells were quantified from three out of 10 consecutive non-overlapping cortical and medullary fields in each kidney under high magnification (n = 3). These three fields were randomly selected in a blinded manner.

After deparaffinization, paraffin sections were placed in citrate-buffered solution (pH 6.0) and boiled for 5 minutes to retrieve antigens. Endogenous peroxidase was quenched with 3.0% hydrogen peroxide in methanol for 20 minutes. Samples were blocked with 3% BSA in PBS for 30 min at room temperature and incubated with primary antibodies (Supplementary Table 1). The sections were labeled with HRP-conjugated secondary antibodies, goat anti-rabbit (ab236469, abcam) or donkey anti-goat (sc-2020; Santa Cruz Biotechnology, Inc. Dallas, TX, USA; 1:500). Diaminobenzidine (DAB) chromogenic substrate (K3468, Agilent Technologies, Inc., Santa Clara, CA) was used for the color reaction, followed by counterstaining with hematoxylin. TUNEL staining for paraffin sections of the mouse kidney at 4 days after the treatments was performed using the ApopTag® Peroxidase In Situ Apoptosis Detection Kit (Merck S7100, Darmstadt, Germany) according to the manufacturer’s protocol. All sections were observed using the Eclipse E600 microscope (Nikon Corporation, Tokyo, Japan). pATM-, pATR-, γH2AX-, KIM-1-, MLH1-, and TUNEL-positive cells were quantified from five of 25 consecutive non-overlapping cortical fields in each kidney under high magnification (n = 5). These five fields were randomly selected in a blinded manner.

Histological sections were subject to antigen retrieval in 60 °C citrate buffer. Primary antibodies against TNF-α (1:100; ab6671; Abcam, Cambridge, UK), IL-6 (1:200; NB600-1131; Novus Biologicals, Littleton, Colorado, USA), IL-10 (1:100; ARC0102, Invitrogen, Waltham, MA, USA), total p38 (1:200; NBP2-19662; Novus Biologicals), and phosphorylated p38 (p-p38) (1:100; GTX59567; GeneTex, Eching, Germany) were applied to the sections and incubated overnight at 4 °C. For negative control, primary antibodies were replaced by an isotope control antibody (IgG; GeneTex). All other steps followed the manufacturer’s instructions (ab236469; Abcam), and all specimens were processed following the identical procedures. Finally, the sections were counterstained by hematoxylin and images were captured on a Leica microscope system (DMRXA2, Leica Microsystems GmbH, Wetzlar, Germany). Quantitative analysis of the positively stained area was performed at the bony callus and compared with the negative control. Expressions of the target protein were quantified by color threshold in ImageJ [5 (link)].