Anti kim 1

Cisplatin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-CD4 (RM4-5) APC/FITC/Pacific blue and anti-TCRβ (H57-597) FITC/BV421, CD45 (30-F11) APC-Cy7/BUV395, anti-CD8α (53 ± 6.7) PerCP-Cy5.5/APC-fire750, were purchased from BD Biosciences. Biotin-conjugated anti-CD16/32 (2.4G2) was purchased from BioLegend. Anti-AGTR2, anti-TIMP-1, and anti-KIM-1 antibodies were purchased from R&D systems (MN, USA). Anti-GAPDH and anti-Bcl-2 antibodies were from Santa Cruz Biotechnology, Inc. (CA, USA). Anti-calbindin antibody was purchased from Cell Signaling Technology, Inc. (MA, USA).

The paraffin-embedded sections were deparaffinized before staining. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide for 10 min before treatment with antibodies of anti-CD36 (Taiclone, Taipei, Taiwan), anti-KIM-1 (R&D Systems, Minneapolis, MN, USA) and anti-SREBP (Abcam, Cambridge, UK). Thereafter, horseradish peroxidase recombinant fusion protein anti-rabbit IgG (1:500) was applied. Finally, DAB chromogen (ScyTeck Laboratories, West Logan, UT, USA) was used and images were acquired using a fluorescent microscope (IX81, Olympus, Tokyo, Japan).

Immunohistochemistry was performed in 1‐μm‐thick paraffin sections of methyl Carnoy's‐fixed specimens using the Vectastain Avidin/Biotin System (Vector Laboratories, Burlingame, CA), as described before 25. Renal tissues were stained using the following primary antibodies: anti‐human Col1A1 (Southern Biotech, Birmingham, AL), anti‐Kim1 (R&D systems, USA), anti‐mouse F4/80 (Serotec, Düsseldorf, Germany), anti‐proliferating cell nuclear antigen (PCNA) (Leinco Technologies, St. Louis, MO) and anti‐Ly6G (BD Biosciences, San Jose, CA). Negative controls for the immunohistochemical procedures consisted of substitution of the primary antibody with non‐immune IgG. Quantification of Ly6G was described before 4. Total number of PCNA‐stained nuclei in renal tubuli was counted in 25 randomly selected fields at ×200 magnification. For staining quantification of collagen and F4/80, 20 consecutive images of renal cortex were taken per section. The percentage of the positively stained area was extracted for intensity using ImageJ software (Wayne Rasband, NIH) and a mean area was calculated. Periodic acid–Schiff (PAS) staining was performed as described before 25, and the tubular injury was scored on a scale of 0–4: 0 = none; 1 = 0–25%; 2 = 25–50%; 3 = 50–75%; 4 = more than 75%. The total score is the calculated average of all tubular scores.