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).
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).