Irrelevant isotype matched and concentration matched rabbit igg

MVECs were seeded onto glass coverslips, grown to 70% confluence, fixed with 3.7% buffered paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Slides were then washed, treated with 3% H₂O₂ in PBS for 15 minutes at room temperature and subsequently blocked with Ultra V block (UltraVision Large Volume Detection System Anti-Polyvalent, HRP; LabVision) for 10 minutes. Cells were incubated overnight at 4 °C with rabbit monoclonal anti-human α-Klotho antibody (catalog number ab181373; Abcam) at 1:20 dilution in 1% BSA in PBS, followed by incubation with biotinylated secondary antibodies and streptavidin peroxidase (UltraVision Large Volume Detection System Anti-Polyvalent, HRP; LabVision) at room temperature.
Immunoreactivity was developed with 3-amino-9-ethylcarbazole (AEC kit; LabVision). Irrelevant isotype-matched and concentration-matched rabbit IgG (Sigma-Aldrich) were used as negative controls. Nuclei were counterstained with Mayer’s hematoxylin (Bio-Optica). Immunolabeled cells were examined with a Leica DM4000 B microscope (Leica Microsystems) and photomicrographs were captured with a Leica DFC310 FX 1.4-megapixel digital colour camera equipped with the Leica software application suite LAS V3.8 (Leica Microsystems).

FLS were seeded onto glass coverslips, grown to 70% confluence and serum-starved for 24 hours. In some experimental points, FLS were subsequently incubated for 24 hours in RPMI 1640 medium with 0.2% FCS containing TNF-α (0.1 ng/ml; PeproTech, Rocky Hill, NJ, USA) and IL-1β (15 pg/ml; PeproTech), alone or in combination. At the end of the experiments, cells were fixed with 3.7% buffered paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Slides were washed with PBS and blocked with 1% BSA in PBS for 1 hour at room temperature, and were then incubated overnight at 4 °C with rabbit polyclonal anti-human AT2R antibody (catalog number ab19134; Abcam) at 1:500 dilution in 1% BSA in PBS, followed by incubation for 45 minutes at room temperature in the dark with Alexa Fluor-488-conjugated secondary antibodies at 1:200 dilution (Invitrogen). Irrelevant isotype-matched and concentration-matched rabbit IgG (Sigma-Aldrich) were used as negative controls. Nuclei were counterstained with DAPI. Immunolabeled cells were examined with a Leica DM4000 B microscope (Leica Microsystems) and fluorescence images were captured with a Leica DFC310 FX 1.4-megapixel digital colour camera (Leica Microsystems).