Aformaldehyde and embedded in paraffin. Tissue was cut at 4 mm and stained with hematoxylin, PAS, and Masson’s trichrome. The degree of glomerulosclerosis and interstitial fibrosis were measured using Image J software (http://rsb.info.nih.gov/ij/). The percentage of glomerulosclerosis was calculated by dividing the total area of PAS positive staining in the glomerulus by the total area of the glomerulus. Interstitial fibrosis was quantified by dividing the area of trichrome stained interstitium by the total cortical area. The mean value of 20 randomly selected glomeruli or five cortical fields was determined 1531364 for each section. Five sections were selected from each kidney.Antigen RetrievalParaffin tissue sections (4 mm) were incubated at 60uC overnight before dewaxing with 2 changes of xylene and 100 ethanol. Tissue sections were immersed in sodium citrate buffer (10 mM sodium citrate, pH 6.0) and heated up in a pressurized cooker to 100uC for 10 minutes. Tissue sections were cooled down to room temperature and prepared for standard immunofluorescence staining procedure.Confocal MicroscopyRenal sections were blocked with PBS containing 1 BSA and incubated with rabbit anti-synaptopodin (1:800) (Sysy antibody, Germany) or rat anti-CD31 (1:100) overnight at 4uC. SectionsGlomerular Endothelial Cell InjuryFigure 5. Apoptosis in glomerular endothelial cells and podocytes in ADR-induced nephropathy in C57BL/6 mice with eNOS deficiency. Apoptotic glomerular endothelial cells (A B) and podocytes (D E), triple labeled with terminal deoxynucleotidyl transferase-mediated digoxigenin-dNTP nick end-labelling (TUNEL; A, B, D and E, green), anti-CD31 (A B, red) and anti-synaptopodin (D E, red), were detected at days 3 (B) and 7 (E) after ADR injection in eNOS-deficient mouse kidneys. Positive apoptotic cells (B E) were counterstained with DAPI nuclear staining. Sections from NS-treated GW-0742 web kidneys (A D) were used as controls. Quantification of CD31+/TUNEL+ glomerular endothelial cells (C) and synaptopodin+/TUNEL+ podocytes in glomeruli (F). Original magnification, 600 X. Magnification in insets, 12006. One-way ANOVA, n = 5, data are means 6 SD. ***: vs NS day 28, P,0.001. doi:10.1371/journal.pone.0055027.gwere probed with goat anti-rabbit or goat anti-rat with Alexa Fluor 555 conjugate (1:2000; Molecular Probes, Eugene, OR). Sections were counterstained with 4, 6-diamidino-2 phenylindole (DAPI) to visualize nuclei and mounted with Fluorescence Mounting Medium (Dako Cytomation). Sections were analyzed with an Pentagastrin biological activity Olympus Fluoview 1000 confocal microscope (Olympus, Tokyo, Japan), FV10-ASW software (version 1.3c; Olympus), oil UPLFL 60x objective (NA1.25; Olympus) at x2 or x3 digital zoom. Contrast and brightness of the images were adjusted further in ImageJ.TUNEL AssayApoptotic assays were performed by TdT mediated X-dUTP nicked labeling (TUNEL) reaction using ApopTagH Fluorescein In Situ Apoptosis Detection Kit (Merck Millipore, Kilsyth, Vic, Australia). Apoptotic endothelial cells and podocytes were identified by double labelling using TUNEL and anti-CD31 or anti-synaptopodin. Goat anti-rat Alexa Fluor 555 conjugate (1:2000) and goat anti-rabbit Alexa Fluor 555 conjugate (1:2000) were used. Sections were counterstained with DAPI.SDS-PAGE gel before transferring to a PVDF membrane. After blocking for 30 minutes at 4uC in blocking buffer (5 skim milk powder in PBS with 0.1 Tween 20), the membrane was incubated overnight with rabbit anti-synaptopodin (1:8000) o.Aformaldehyde and embedded in paraffin. Tissue was cut at 4 mm and stained with hematoxylin, PAS, and Masson’s trichrome. The degree of glomerulosclerosis and interstitial fibrosis were measured using Image J software (http://rsb.info.nih.gov/ij/). The percentage of glomerulosclerosis was calculated by dividing the total area of PAS positive staining in the glomerulus by the total area of the glomerulus. Interstitial fibrosis was quantified by dividing the area of trichrome stained interstitium by the total cortical area. The mean value of 20 randomly selected glomeruli or five cortical fields was determined 1531364 for each section. Five sections were selected from each kidney.Antigen RetrievalParaffin tissue sections (4 mm) were incubated at 60uC overnight before dewaxing with 2 changes of xylene and 100 ethanol. Tissue sections were immersed in sodium citrate buffer (10 mM sodium citrate, pH 6.0) and heated up in a pressurized cooker to 100uC for 10 minutes. Tissue sections were cooled down to room temperature and prepared for standard immunofluorescence staining procedure.Confocal MicroscopyRenal sections were blocked with PBS containing 1 BSA and incubated with rabbit anti-synaptopodin (1:800) (Sysy antibody, Germany) or rat anti-CD31 (1:100) overnight at 4uC. SectionsGlomerular Endothelial Cell InjuryFigure 5. Apoptosis in glomerular endothelial cells and podocytes in ADR-induced nephropathy in C57BL/6 mice with eNOS deficiency. Apoptotic glomerular endothelial cells (A B) and podocytes (D E), triple labeled with terminal deoxynucleotidyl transferase-mediated digoxigenin-dNTP nick end-labelling (TUNEL; A, B, D and E, green), anti-CD31 (A B, red) and anti-synaptopodin (D E, red), were detected at days 3 (B) and 7 (E) after ADR injection in eNOS-deficient mouse kidneys. Positive apoptotic cells (B E) were counterstained with DAPI nuclear staining. Sections from NS-treated kidneys (A D) were used as controls. Quantification of CD31+/TUNEL+ glomerular endothelial cells (C) and synaptopodin+/TUNEL+ podocytes in glomeruli (F). Original magnification, 600 X. Magnification in insets, 12006. One-way ANOVA, n = 5, data are means 6 SD. ***: vs NS day 28, P,0.001. doi:10.1371/journal.pone.0055027.gwere probed with goat anti-rabbit or goat anti-rat with Alexa Fluor 555 conjugate (1:2000; Molecular Probes, Eugene, OR). Sections were counterstained with 4, 6-diamidino-2 phenylindole (DAPI) to visualize nuclei and mounted with Fluorescence Mounting Medium (Dako Cytomation). Sections were analyzed with an Olympus Fluoview 1000 confocal microscope (Olympus, Tokyo, Japan), FV10-ASW software (version 1.3c; Olympus), oil UPLFL 60x objective (NA1.25; Olympus) at x2 or x3 digital zoom. Contrast and brightness of the images were adjusted further in ImageJ.TUNEL AssayApoptotic assays were performed by TdT mediated X-dUTP nicked labeling (TUNEL) reaction using ApopTagH Fluorescein In Situ Apoptosis Detection Kit (Merck Millipore, Kilsyth, Vic, Australia). Apoptotic endothelial cells and podocytes were identified by double labelling using TUNEL and anti-CD31 or anti-synaptopodin. Goat anti-rat Alexa Fluor 555 conjugate (1:2000) and goat anti-rabbit Alexa Fluor 555 conjugate (1:2000) were used. Sections were counterstained with DAPI.SDS-PAGE gel before transferring to a PVDF membrane. After blocking for 30 minutes at 4uC in blocking buffer (5 skim milk powder in PBS with 0.1 Tween 20), the membrane was incubated overnight with rabbit anti-synaptopodin (1:8000) o.
