The anatomopathological

samples were analysed by a pathologist blind to group assignments. The kidneys were fixed in a 10% neutral buffered formalin solution, embedded in paraffin and used for histopathological examination. learn more Four micrometres-thick sections were cut, deparaffinized, hydrated and stained with haematoxylin and eosin (H&E). The renal sections were examined in a blinded fashion for grade of cortical tubular epithelial necrosis. Counts were performed in at least 10 different fields of square micrometres and assigned for severity of necrosis, using scores on a scale of 1 (<5%), 2 (5–25%), 3 (25–50%), 4 (50–75%) and 5 (>75%) [23]. TUNEL assay was performed according to the manufacturer’s instructions (Apoptag; Oncor, Gaithersburg, MD, USA). Briefly, deparaffinized 4 µm-thick sections of paraffin-embedded tissues were pretreated with 20 µl/ml Proteinase K (Dako, Glostrup, Denmark) for 30 min at 37°C. EPZ015666 purchase After washing, sections were incubated with digoxygenin-labelled dUTP in the presence of terminal deoxynucleotidyl transferase. After the enzymatic reaction was blocked, sections were incubated with a specific peroxidase-labelled anti-digoxin antibody. Peroxidase was then reduced by 0·05 diaminobenzidine (Sigma, St Louis, MO, USA) in 0·1 ml/l phosphate-buffered saline (PBS), pH 7·6 containing 1% H2O2. After washing, the sections were lightly stained

with haematoxylin. Negative control reactions were performed for each reaction step. They were obtained by omission of terminal deoxynucleotidyl transferase, anti-digoxin antibody and peroxidase substrate. Positive controls included sections of paraffin-embedded

lymphoma of human origin. The external medullar region was examined and the total number of labelled nuclei was counted. Ten fields of 1 mm2 were examined by means of a reticulated lens. Sections from 4 µm thick were applied to poly-2-lysine coated slides. Sections were dewaxed in xylene, dehydrated through graded alcohols and water and then immersed in 0·3% vol/vol H2O2 in methanol for 30 min to block endogenous peroxidase. Antigens were reduced by microwaving at 750 W for 15 min in 0·01 mol/l trisodium citrate buffer, pH 6·0, then rinsed well in standard PBS and non-specific binding was blocked with 10% equine serum in PBS. Sections were incubated with primary antibodies of monoclonal origin against C3 (clone B-9) or with polyclonal from goat against TNF-α, interleukin (IL)-6 and Bcl2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After being rinsed with PBS, sections were incubated with biotinylated secondary antibodies. Afterwards, sections were rinsed with PBS and incubated with avidin–biotin horseradish peroxidase complex according to the manufacturer’s instructions (Vectastain Universal Quick Kits; Vector Laboratories Ltd, Peterborough, UK).