Co-localization of NKA α1 and Lyso-Tracker Red was performed by immunocytochemistry as previously described.33 (link) PTECs were incubated with Lyso-Tracker Red diluted 1:1000 for 30 min at 37 °C. Next, the cells were rinsed three times with PBS and fixed in 2% formaldehyde/150 mM NaCl/20 mM Hepes (pH, 7.8) for 30 min at room temperature. After quenching with 50 mM Tris in staining buffer (0.1% Triton X-100/100 mM NaCl/20 mM Hepes pH, 7.8) for 1 h and rinsing with staining buffer, the cells were then incubated overnight at 4 °C with a mouse monoclonal anti-NKA α1 antibody diluted 1:200 in staining buffer. After rinsing in staining buffer, the cells were incubated for 1 h at room temperature with fluorescein isothiocynate-conjugated goat anti mouse secondary antibody diluted 1:500 in staining buffer. Fluorescence was detected using an LSM510 Meta Confocal laser-scanning microscope (Leica, TCS-SP5, Solms, Germany).
Renal tissue immunohistochemistry was performed on 4 μm sections of formaldehyde-fixed and paraffin-embedded renal cortices of rats. Primary antibodies against NKA α1 (1:200) were incubated at 4 °C overnight after the retrieval of antigens, followed by reacting with horseradish peroxidase-labeled secondary antibody (1:200) at 37 °C for 1 h. The color reaction was induced by 3,3'-diaminobenzidine, followed by counterstaining with 10% Mayer’s hematoxylin.
Renal tissue immunohistochemistry was performed on 4 μm sections of formaldehyde-fixed and paraffin-embedded renal cortices of rats. Primary antibodies against NKA α1 (1:200) were incubated at 4 °C overnight after the retrieval of antigens, followed by reacting with horseradish peroxidase-labeled secondary antibody (1:200) at 37 °C for 1 h. The color reaction was induced by 3,3'-diaminobenzidine, followed by counterstaining with 10% Mayer’s hematoxylin.