The intra abdominal temperature was main tained at 36 0. 1 C with a heating pad which was servo adjusted by a temperature controller throughout the experiment. For sur vival experiments, mice have been monitored on a every day basis with a scoring assay depending on physique weight, activity and common appearance as reported previously. Any animals that scored 7 have been euthanized. All animals received 0.5 ml saline i. p. injection per every six hrs for the very first 24 hrs right after experiments. Immunoblot Proteins have been extracted from treated HK2 cells lines or frozen kidney samples by cell disruption in cell lysis buf fer and sonication with an ultrasonic probe, followed by centrifugation at 10,000 g for ten minutes at four C. The supernatant was collected for Western blotting.
Samples containing 30 ug of extracted protein, as determined by the Bradford Motesanib AMG-706 protein assay, have been loaded on a NuPAGE 4 to 12% Bis Tris gel for protein fractio nation by electrophoresis after which electro transferred to a nitrocellulose membrane. Blots were blocked with 5% non fat dry milk in TBS, and probed with appropriate antibodies by HRP conjugated secondary antibodies and visualisation with enhanced chemiluminescence. a tubulin was applied as internal handle. Densitometry analysis had been preformed and normalized using a tubulin after which pre sented as percentage of control. Histologic score The sum score was calculated from the evaluation of 10 cor tical tubules cross section stained with H E by utilizing a modified scoring program, 0, no harm.
1, mild harm, rounded epithelial cells and dilated tubular lumen, two, moderate harm, selleck inhibitor flattened epithelial cells, loss of nuclear staining and substantially dilated lumen, three, extreme harm, destroyed tubules with no nuclear staining of epithelial cells. Immunohistochemistry The second l death of tubular epithelial cells was detected by in situ TUNEL assay Obiogene, Cambridge, UK according to the companies guidelines. The fixed cryostat sections have been washed with PBS then treated with proteinase K at room temperature for 15 minutes. For optimistic controls, sections had been treated with nuclease at 37 C for 15 minutes. The sections were quenched in 3% hydrogen peroxide in PBS for five minutes. The quenched sections have been labelled with TDT enzyme at 37 C for 1 hour within a humidified chamber and subsequently incubated with anti digoxygenin conjugated to horseradish peroxidase for 30 minutes at room temperature.
They were then stained with DiAminoBenzidine. The sum of the TUNEL cells in an objective grid from ten places of randomly chosen renal cortex was counted below a 40 objective lens by an investigator who was blinded for the experimen tal protocol. The other fixed cryostat sections had been incubated with 3% hydrogen peroxide for 30 minutes to quench endogen ous peroxidase activity.