Ected using PE-conjugated goat anti-rabbit and FITCconjugated goat anti-mouse secondary antibody for 2 h in the dark. Finally, CD34+/Flk+ protein expression was analyzed and evaluated with confocal laser scanning microscopy.Flow CytometryFor quantification of EPCs in ischemic kidneys using flow cytometry, mononuclear cells were isolated by a mechanical process. Kidney tissue (about 100 mg) was homogenized with 2 ml PBS in a glass homogenizer and then 
filtered through a 200 mm nylon mesh (BD Biosciences; San Jose, CA, USA). Single-cell suspensions were centrifuged at 2000 g for 5 min, resuspended with 22948146 2 ml PBS, and then incubated for 30 min with a rabbit polyclonal anti-Flk-1 antibody (Novus Biologicals; Littleton, CO, USA) and FITC-conjugated mouse monoclonal anti-CD34 antibody (Santa Cruz Biotechnology) at room temperature. The secondary antibody for Flk-1 staining was goat anti-rabbit IgG-PE (Santa Cruz Biotechnology). After washing, immunofluorescence was detected using flow cytometry. To quantify EPCs, the number of CD34/Flk-1 double-positive cells within the mononuclear cell population was counted.Histological ExaminationFormalin-fixed tissues were embedded in paraffin and sectioned at 5 mm and stained with haematoxylin and eosin (H E). The sections were examined microscopically by an experienced pathologist who was blinded to the treatment that each animal received. Renal injury was defined as 4EGI-1 biological activity tubular necrosis, tubular dilatation and/or atrophy, inflammatory cell infiltration, cellular edema, or tubule cast formation [16], with a scoring range from Grade 0 to 4. Higher scores represent more severe damage: 0, normal kidney; 1, minimal necrosis (,25 involvement of the cortex or outer medulla); 2, mild necrosis (25?0 involvement of the cortex or medulla); 3, moderate necrosis (50?5 involvement of the cortex or medulla); and 4, severe necrosis (.75 involvement of the cortex or medulla).Immunohistochemical StainingPeritubular capillary rarefaction index (PCRI) was analyzed for peritubular capillary densities using a monoclonal antibody to CD34 and stained using immunohistochemistry. To determine cell proliferation, immunohistochemical staining with proliferating cell nuclear antigen (PCNA) was performed. Briefly, paraffin-embedded blocks were sectioned at a 5 mm, dewaxed and rehydrated. Antigens were retrieved with microwave pretreatment in citrate buffer (pH 6.0). Endogenous peroxidase was blocked with 3 H2O2 for 15 min, and then nonspecific NHS-Biotin web binding sites were blocked with 4 goat serum diluted 1:10 in PBST (PBS, pH 7.4, 0.05 1516647 Tween 20). Sections were incubated with a rabbit anti-CD34 antibody (ABbiotec, San Diego, 
CA, USA) at 1:100 dilution or rabbit anti-PCNA antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1:100 dilution overnight at 4uC. Primary antibodies were detected with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody and developed with 3,39diaminobenzidine tetrahydrochloride. Negative controls were performed by omitting the primary antibody. Finally, PCRI was determined using the procedure of Kang et al [17], and the number of PCNA-positive cells were counted in 10 nonoverlapping sequential fields at 4006magnification [18].q-PCR AnalysismRNA expression of VEGF-A, stromal cell-derived factor-1a (SDF-1a) and insulin like growth factor 1 (IGF-1) was assessed using qPCR. Total RNA was extracted from renal tissue with Trizol reagent (Invitrogen; Carlsbad, CA, USA). Total RNA concentration was.Ected using PE-conjugated goat anti-rabbit and FITCconjugated goat anti-mouse secondary antibody for 2 h in the dark. Finally, CD34+/Flk+ protein expression was analyzed and evaluated with confocal laser scanning microscopy.Flow CytometryFor quantification of EPCs in ischemic kidneys using flow cytometry, mononuclear cells were isolated by a mechanical process. Kidney tissue (about 100 mg) was homogenized with 2 ml PBS in a glass homogenizer and then filtered through a 200 mm nylon mesh (BD Biosciences; San Jose, CA, USA). Single-cell suspensions were centrifuged at 2000 g for 5 min, resuspended with 22948146 2 ml PBS, and then incubated for 30 min with a rabbit polyclonal anti-Flk-1 antibody (Novus Biologicals; Littleton, CO, USA) and FITC-conjugated mouse monoclonal anti-CD34 antibody (Santa Cruz Biotechnology) at room temperature. The secondary antibody for Flk-1 staining was goat anti-rabbit IgG-PE (Santa Cruz Biotechnology). After washing, immunofluorescence was detected using flow cytometry. To quantify EPCs, the number of CD34/Flk-1 double-positive cells within the mononuclear cell population was counted.Histological ExaminationFormalin-fixed tissues were embedded in paraffin and sectioned at 5 mm and stained with haematoxylin and eosin (H E). The sections were examined microscopically by an experienced pathologist who was blinded to the treatment that each animal received. Renal injury was defined as tubular necrosis, tubular dilatation and/or atrophy, inflammatory cell infiltration, cellular edema, or tubule cast formation [16], with a scoring range from Grade 0 to 4. Higher scores represent more severe damage: 0, normal kidney; 1, minimal necrosis (,25 involvement of the cortex or outer medulla); 2, mild necrosis (25?0 involvement of the cortex or medulla); 3, moderate necrosis (50?5 involvement of the cortex or medulla); and 4, severe necrosis (.75 involvement of the cortex or medulla).Immunohistochemical StainingPeritubular capillary rarefaction index (PCRI) was analyzed for peritubular capillary densities using a monoclonal antibody to CD34 and stained using immunohistochemistry. To determine cell proliferation, immunohistochemical staining with proliferating cell nuclear antigen (PCNA) was performed. Briefly, paraffin-embedded blocks were sectioned at a 5 mm, dewaxed and rehydrated. Antigens were retrieved with microwave pretreatment in citrate buffer (pH 6.0). Endogenous peroxidase was blocked with 3 H2O2 for 15 min, and then nonspecific binding sites were blocked with 4 goat serum diluted 1:10 in PBST (PBS, pH 7.4, 0.05 1516647 Tween 20). Sections were incubated with a rabbit anti-CD34 antibody (ABbiotec, San Diego, CA, USA) at 1:100 dilution or rabbit anti-PCNA antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1:100 dilution overnight at 4uC. Primary antibodies were detected with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody and developed with 3,39diaminobenzidine tetrahydrochloride. Negative controls were performed by omitting the primary antibody. Finally, PCRI was determined using the procedure of Kang et al [17], and the number of PCNA-positive cells were counted in 10 nonoverlapping sequential fields at 4006magnification [18].q-PCR AnalysismRNA expression of VEGF-A, stromal cell-derived factor-1a (SDF-1a) and insulin like growth factor 1 (IGF-1) was assessed using qPCR. Total RNA was extracted from renal tissue with Trizol reagent (Invitrogen; Carlsbad, CA, USA). Total RNA concentration was.
