This difference could be attributed to differences in dosing regimens between the two studies

-containing regions were taken using a Zeiss Axioscop microscope with AxioVision image capture software at a magnification of 400x. On these photos the glomeruli were defined PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19683258 as the area of interest. These aoi were isolated using the image j software. In the next step the Relebactam site nuclei in the aoi were counted using a semiquantitative computer assisted method. Then the stained area of each aoi was determined. Briefly, the image j program offers a colour deconvolution plug which splits images into three color-channels and is widely used for immunohistochemistry analysis. The number of nuclei could be calculated after the hematoxilin stained areas were isolated using the “H&E”filter of the color deconvolution plug in. After that “watershed”and “particle counter”algorithms were run for the nuclei count as described by others. This method was compared with manual counting and was found to be as accurate as the manual method. For the determination of the stained area we used the colour deconvolution plug in with the RGB filter. The red image was used for the calculation of the stained area. For the determination of the background for the stained area determination the red dye intensity from a control which was treated only with the secondary antibody and the red dye was set as threshold for the filter settings for the image PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19684114 j analysis. The ratio of stained area/nuclei was calculated and the statistical analysis was performed as further described. 5. VEGF-A plasma levels Blood samples of all monkeys were collected in tubes containing EDTA before intravitreal injection and on day one and seven after injection of anti- 6 / 20 Renal Effects of Intravitreally Injected Anti-VEGF Agents VEGF agents, and plasma was prepared by centrifugation, transferred to new tubes and stored at 270 C. Plasma samples were analyzed using commercially available ELISA kits for human VEGF-A . Briefly, the microtitration plates were coated with monoclonal antibodies specific for VEGF-A, standards and probes were added, incubated and washed. Then, an enzyme-linked polyclonal antibody specific for VEGF-A was added and its substrate solution followed after a second incubation and wash step. After stopping the colour development, the intensity of colour was measured by photospectrometry with the lower detection limit of VEGF-A set at 30 pg/ml. Calculation of VEGF concentration was performed according to the manufacturer’s recommendations. 6. Light and electron microscopy Specimens were postfixed with 1% OsO4 at room temperature in 0.1 M cacodylate buffer, en bloc stained with uranyl acetate and lead citrate, and embedded in Epon after dehydration in a graded series of acetones. Semithin sections were stained with Toluidine Blue and examined by light microscopy 7 / 20 Renal Effects of Intravitreally Injected Anti-VEGF Agents . For electron microscopy, the sections were cut ultrathin and analyzed with a Zeiss 902 A electron microscope. 7. Quantification of the glomerular endothelial fenestrations Under the light microscope at a low magnification glomeruli were identified in semi-thin sections and checked on mechanical or fixation artifacts and pathological features at a higher magnification. Three glomeruli per kidney with middle to large diameter and intact bowman’s capsules were chosen and cut ultrathin for electron microscopy. After examination of the probes at a magnification of 3000 fold, montages of transmission electron micrographs were performed by using the multiple image a