Facility line

Paraffin sections were antigen-retrieved by high-pressure cooking in 0.1 M citrate buffer pH=6.0, followed by blocking endogenous peroxidase with 3% H₂O₂ in 100% methanol and by a blocking step using 5% skim milk/PBS. Sections were incubated with the respective primary antibody overnight followed by the suitable HRP- or fluorescence-coupled secondary antibody (Dianova). Signals were generated using 3,3’-diaminobenzidine for HRP staining. Specimens were analyzed using the Leica Axiovert microscope or STED microscope (FACILITY Line, Abberior).
Five-micrometer cryo sections were antigen-retrieved using 0.5% tritonX-100/PBS, blocked with 5% skim milk/PBS, incubated overnight at 4 °C with either acetylated tubulin, nNOS/NOS1, klotho, or glucocorticoid receptor followed by incubation with a suitable fluorochrome-coupled secondary antibody (Dianova). Nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole, 100 ng/mL).
Cells grown on glass coverslips were fixed with 4% paraformaldehyde (PFA)/PBS for 10 min, permeabilized with 0.5% tritonX-100/PBS for 30 min, and blocked with 5% skim milk/PBS for 1 h. For detection of cell borders, cells were stained with ZO-1 followed by incubation with anti-ZBTB16 (Sigma; cat.no. HPA001499) and suitable fluorochrome-coupled secondary antibody (Dianova). Samples were analyzed using a STED microscope (FACILITY Line, Abberior).

Cell proliferation was assessed using antigen-retrieved paraffin sections co-stained with an antibody against the proliferation marker Ki-67 and DAPI. Images were captured using Abberior Facility Line (Abberior Instruments GmbH, Göttingen, Germany). Channels were merged, and signal was evaluated using Fiji/ImageJ software. The number of Ki-67–positive epithelial nuclei per visual field (40× magnification) [49 (link),50 (link)] were counted. About ten randomly chosen areas in renal cortex were analyzed.

Cells were seeded on coverslips or glass-bottomed dishes and cultured to a suitable density (24 h) at 37 °C in a 5% CO₂ atmosphere with 95% humidity. Cells were incubated with HBmito Crimson for 10 min under the same conditions, after which images were obtained using a confocal laser-scanning microscope. STED imaging was performed using the Abberior Facility Line (Abberior Instruments GmbH, Germany) with a 637-nm laser for excitation and a 775-nm pulsed laser for STED depletion. A 60× oil-immersion objective (N.A. 1.42, Olympus, Japan) was employed in imaging experiments. All STED results presented in the paper have been processed by Huygens deconvolution software. The deconvolution parameter is set to improve the resolution while maintaining the real structure. The acquisition parameters of the microscope are shown in Table S2.