Las v3

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).

The length of primary root was measured every day to calculate the average increment during the whole treatment period. After 7 days of treatment, cucumber roots were cut off and photographed. Root cell viability was then assessed by fluorescein diacetate (FDA)–propidium iodide (PI) double staining method as described by Bose et al. (2014 (link)). Root segments, including the root tip (1 cm), were cut off and stained with 5 μg mL⁻¹ FDA for 3 min followed by 3 μg mL⁻¹ PI for 10 min. Then the stained roots were observed using a Leica DM2500 microscope (Leica Microsystems, Wetzlar, Germany). Excitation wavelengths of 488 and 594 nm were used for FDA and PI imaging, respectively. Images were acquired with a digital camera (Leica DFC495, Leica Microsystems) equipped with a LAS V3.8 (Leica Microsystems) software.

The blood smears were fixed with absolute methanol and stained with 10% Giemsa Methylene Blue Eosin Merck^® diluted in distilled water (pH 7.0 for 50 min), according to Eisen and Schall [26 (link)], at the Parasitology division from UNESP, Botucatu. For morphological analysis of the intra-erythrocytic parasite stages, digital images were captured and measured using a compound microscope at 1000× magnification with the Leica software application suite LAS V3.8 (Leica Microsystems). Measurements are in micrometres (μm) comprising the parasite’s length and width, with mean and standard deviation (means ± standard deviation) given. Parasitaemia was calculated per 100 erythrocytes, with ~10⁴ erythrocytes examined per blood smear following Cook et al. [16 (link)].

Both EC-injured and control EDL muscles (n = 5 each) were fixed with 10% formalin in PBS, dehydrated with a graded alcohol series, cleared in xylene, and embedded in paraffin. Muscles sections (5 μm thick) were deparaffinized and routinely stained with hematoxylin and eosin. Tissue morphology was then observed under a light microscope (Leica DM4000 B) equipped with a DFC310 FX 1.4-megapixel digital color camera and the software application suite LAS V3.8 (Leica Microsystems, Mannheim, Germany).

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.

Gastric fundus muscle samples were embedded in paraffin (n = 3 each) after being fixed with 10% formalin in phosphate-buffered saline (PBS), dehydrated with a graded alcohol series and cleared in xylene. From each sample (n = 30 for each experimental condition), at least 10 sections (thickness 5 µm) were sliced and colored with H&E. Morphological analysis of the tissue was achieved by a light microscope (Leica DM4000 B) equipped with a DFC310 FX 1.4-megapixel digital color camera and software application suite LAS V3.8 (Leica Microsystems, Mannheim, Germany).