Then, these signaling pathways are inactivated by Necl-4

ed under diethyl Light Microscopic Study Tissues fixed in 2% PBS were embedded in paraffin using routine protocols. Paraffin-embedded materials were sectioned at 1-mm thickness for routine staining with periodic acid-Schiff and Masson trichrome. Luminal hyaline casts were assessed in 20 fields for each PAS-stained section. The number of casts was counted under x 200 magnification, and the mean number per field was calculated. For evaluating renal tubular injury, 25 fields from each Masson trichrome-stained section were evaluated under x200 magnification. The extent of tubular injury was assessed by counting the percentage of areas with tubular dilatation, tubular cast formation, and tubular epithelial cell necrosis per field of cortex. Scores from 0 to 5 were used: 0, none; 1,,10%; 2, 10 25%; 3, 2550%; 4, 5075%: 5,.75% of areas injured, and the results were averaged. Immunohistochemistry The antibodies used in this study were as follows: mouse monoclonal anti-rat ED1 antibody as a macrophage marker, mouse monoclonal anti- proliferating cell nuclear antigen antibody 2 Erlotinib Attenuates Cisplatin-Induced Nephrotoxicity as a proliferating cell marker, and rabbit polyclonal anti-active caspase-3 antibody as a marker of apoptosis. EnVisionTM+Dual Link System HRP, based on HRP-labeled polymer, was purchased from Dako. Immunohistochemical staining for ED1, PCNA, and caspase-3 was performed as follows: the paraffin buy Chebulinic acid Sections of renal tissues were dewaxed and washed in PBS. H2O2 was added to slides for 30 min for the quenching of endogenous peroxidase. Sections were washed in PBS, treated with protein blocking solution, and incubated overnight at 4uC with the anti-ED1, anti-PCNA, and anti-active caspase-3 antibodies as the primary antibodies. Sections were pretreated twice for 5 min in a conventional household microwave for ED1 staining or autoclave-heated at 121uC for 10 min for PCNA staining before application of the primary antibodies. Sections were washed thrice in PBS, then incubated with EnVisionTM+Dual Link System-HRP for 40 min at room temperature. Next, the sections were developed using diaminobenzidine as the substrate to produce a brown stain, and sections were counterstained with hematoxylin for ED1 and PCNA staining and with methyl green for caspase-3 staining. Cells undergoing apoptosis PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19690518 were identified by in situ terminal deoxynucleotidyl transferase -mediated dUTP nick end labeling using the ApopTag plus peroxidase in situ apoptosis detection kit. The quantification of PCNA, ED-1, TUNEL, and caspase-3positive cells in the tubulointerstitium was performed by counting the number of positively stained cells PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19692147 in 25 consecutive renal cortical fields under x 400 magnification, and the values were averaged per field. Human Proximal Tubular Cell Culture Human proximal tubular cells that exhibit biochemical and morphological features of normal proximal tubular cells in culture were obtained from the American Type Culture Collection. Cells were grown in DMEM containing 10% fetal bovine serum, a 1% streptomycin-penicillin mixture, 44 mM NaHCO3, and 14 mM HEPES in an atmosphere of 5% CO2 and 95% air at 37uC in a humidified incubator. Experiments were performed with cells up to the fifth passage, as it has been shown that there are no phenotypic changes up to this passage number. Bio-Plex Suspension Array System HK-2 cells were initially cultured for 48 hours in medium containing 10% FBS, and then cultured overnight in medium containing 2% FB