Mitotracker® Red CMXRos (Thermo Fisher Scientific) to visualize mitochondrial networks in live mGECs [34 (link)].
mGECs on coverslips were fixed with methanol and incubated with anti-8-oxoG monoclonal antibody (N45.1; Japan Institute for the Control of Aging) previously described [35 (link)]. Cells were also stained with either goat anti-mtTFA antibodies (Santa Cruz), rabbit anti-LC3 (Novus Biologicals), or rabbit anti-phospho-histone H2A.X (Ser139) Antibody (Cell Signaling Technology). The antigen-antibody complexes were visualized with Alexa Fluor secondary antibodies. DAPI was added to mounting medium. mGECs plated in 12-well slide (Ibidi) at a density of 2.0 × 104 cells/well and were transfected with WT and mutant GFP-endoG and fixed with 4% PFA in PBS. DAPI was added to mounting medium. Podocytes on coverslips were fixed with 4% PFA in PBS, incubated anti-Caspase 3 antibodies (cleaved Santa Cruz) and Phalloidin-FITC (Sigma). DAPI was added to mounting medium. The cells were imaged with a Zeiss Axioplan2 equipped with Q-imaging MP3.3 RTV color camera running QED capture software.
mGECs on coverslips were fixed with methanol and incubated with anti-8-oxoG monoclonal antibody (N45.1; Japan Institute for the Control of Aging) previously described [35 (link)]. Cells were also stained with either goat anti-mtTFA antibodies (Santa Cruz), rabbit anti-LC3 (Novus Biologicals), or rabbit anti-phospho-histone H2A.X (Ser139) Antibody (Cell Signaling Technology). The antigen-antibody complexes were visualized with Alexa Fluor secondary antibodies. DAPI was added to mounting medium. mGECs plated in 12-well slide (Ibidi) at a density of 2.0 × 104 cells/well and were transfected with WT and mutant GFP-endoG and fixed with 4% PFA in PBS. DAPI was added to mounting medium. Podocytes on coverslips were fixed with 4% PFA in PBS, incubated anti-Caspase 3 antibodies (cleaved Santa Cruz) and Phalloidin-FITC (Sigma). DAPI was added to mounting medium. The cells were imaged with a Zeiss Axioplan2 equipped with Q-imaging MP3.3 RTV color camera running QED capture software.
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