Dfc310 fx 1.4 megapixel

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).

dMVECs were grown to confluence on glass coverslips, starved in EBM with 2% FBS overnight and then incubated for 24 hours in EBM containing 2% FBS and 10% of serum from lSSc or dSSc patients, naïve or under pharmacological therapy with CYC, or 10% of serum from healthy controls. dMVECs were subsequently fixed in 3.7% buffered paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. For immunofluorescent detection and quantification of cell apoptosis we used the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technology (Fluorescein Isothiocyanate (FITC) In Situ Cell Death Detection Kit; Roche Diagnostics) according to the manufacturer’s instructions. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). The stained cells were observed under a Leica DM4000 B microscope (Leica Microsystems, Mannheim, Germany) and photographed using a Leica DFC310 FX 1.4-megapixel digital colour camera equipped with the Leica software application suite LAS V3.8 (Leica Microsystems). The percentage of apoptotic dMVEC nuclei was calculated as TUNEL/DAPI-positive nuclei in proportion to all DAPI-positive nuclei. Counting was performed on ten randomly chosen microscopic fields (x40 original magnification) per sample by two independent blinded observers.