Axio imager 1

BMSCs were seeded on a glass coverslip and let to attach at 37°C, 5% CO₂ for 24 h. The cells were fixed with 4% PFA at RT for 15 min, permeabilized with 0.05% Triton X-100 at RT for 5 min and blocked with 10% normal goat serum (Abcam, UK) at RT for 1 h. Primary antibodies were added and incubated at +4°C overnight. The following antibodies and concentrations were used: rabbit anti-CD44 (Abcam, UK; 0.2 μg/ml, ab157107), rabbit anti-CD45 (Merck, USA; SAB4502541, 10μg/ml), and mouse anti-CD90 (Abcam, UK; ab225, 1 μg/ml). Cells were then washed three times with 0.1% Tween-20 and incubated at RT for 1 h with the following secondary antibodies: Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (Abcam, UK; ab150077, 2 μg/ml) and Alexa Fluor^® 594-conjugated goat anti-mouse IgG (Abcam, UK; ab150116, 2 μg/ml). Nuclei were stained with Vectashield Antifade with 1.5 µg/ml DAPI (Vector Laboratories, USA). Stained cells were then imaged with Zeiss Axio Imager 1 (Zeiss, Germany) under standardized imaging setup. Images were processed in Zen Blue 2 (Zeiss) and ImageJ 1.53c software and pseudo-colored cyan (secondary antibodies) and magenta (DAPI).

In addition, the aortic valve peak flow velocity and aortic valve mean gradient were measured in the apical five-chamber view by continuous wave doppler in systole. The presence or absence of aortic regurgitation was visualized in this five-cavity view by color Doppler. Furthermore, aortic stenosis was estimated in two-dimensional parasternal long-axis view by measuring the aortic valve cusp separation in systole.
At sacrifice, the heart was harvested and weighed, and a section of the left ventricle was snap-frozen for subsequent determination of LV fibrosis, using 8 µm thick histological slices stained with Sirius Red as previously described [12 (link)]. In addition, the aortic valve was carefully dissected and incubated over 24 h at 4 °C in a solution of a 20 nM Osteosense 680Ex^® (Perkin-Elmer, Waltham, MS, USA) in PBS. This fluorescent probe binds to hydroxyapatite with high affinity and thus allows for the detection of microcalcifications. Then, the valve was rinsed and snap-frozen for subsequent analysis using 8 µm thick histological slices with mounting medium containing DAPI. Pictures were acquired on an epifluorescence microscope (Axio Imager 1, Zeiss, Jena, Germany) equipped with an apotome using the Cyanine 5.5 filter for calcification detection and a DAPI filter for cell nuclei detection.

C2 and NI‐1 cells were incubated in control medium, vehicle control or in various concentrations of HR1 antagonists (30–125 µM) in 24‐well microtiter plates (5 × 10⁵ cells/well) at 37°C for 24 or 48 hr. After incubation, cells were harvested and cytospin slides were prepared using Cytospin^TM 4 Cytocentrifuge from ThermoFisher Scientific (Waltham, MA, USA). Cytospin slides were stained with the Hematek® Stain Pak (Modified Wright's Stain #10310965) obtained from Siemens Healthineers (Erlangen, Germany) on a Hematek® Slide Stainer obtained from Bayer HealthCare LLC (Leverkusen, Germany) and three visual fields per cytospin slide were quantified by light microscopy. In three visual fields (400× magnification), approximately 200 cells were counted and the percentages of viable, apoptotic and necrotic cells were calculated from total cell numbers. Apoptosis and necrosis were defined according to characteristic morphological features (Van Cruchten & Van Den Broeck, 2002). Apoptosis in drug‐exposed cells was confirmed by the “TUNEL‐In situ cell death detection kit‐fluorescein” kit from Roche (Mannheim, Germany) following the manufacturer's instructions. The fluorescent stain 4′,6‐diamidino‐2‐phenylindole (DAPI; Sigma‐Aldrich) was used to visualize cell nuclei. Images were obtained using Zen imaging software and Axio Imager 1, both from Carl Zeiss (Oberkochen, Germany).

Tissues and explant cultures were fixed with 4% paraformaldehyde (PFA) and embedded in paraffin. The samples were sectioned in 5 μm slices and deparaffinized. The heat-induced antigen retrieval was performed whether with a microwave oven or a pressure cooker in a citrate buffer solution (Dako). Histochemical stainings were carried out using standard techniques for IHC and IHC-IF. Images were taken with a Leica DM LB microscope or with a Zeiss AxioImager 1 (Biomedicum Imaging Unit, University of Helsinki). The list of used antibodies are shown in Supplementary Data 2.

For Western blots, haemolymph samples were collected as follows. Twenty-five female flies were placed on a 10-μM filter of an empty Mobicol spin column (Mobitec, Goettingen, Germany), covered with glass beads and centrifuged for 20 min at 4 °C, 10,000 g into a tube containing 50 μL of PBS supplemented with complete protease inhibitor solution (Roche) and 1 mM phenylmethylsulfonyl fluoride. The protein concentration of the samples was determined by Bradford assay, and 30 μg of protein extract was separated on a 4–12% acrylamide precast Novex gel (Invitrogen) under reducing conditions and transferred to nitrocellulose membranes (Invitrogen iBlot). After blocking in 5% non-fat dry milk in PBS containing 0.1% Tween-20 for 1 h, membranes were incubated at 4 °C overnight with a mouse anti-green fluorescent protein (GFP) antibody (Roche) in a 1:1500 dilution, or a rabbit anti-lipophorin antibody (kind gift of Dr. Suzanne Eaton) in a 1:1000 dilution. Donkey anti-mouse-horseradish peroxidase (HRP) or anti-rabbit-HRP secondary antibody (Dako) in a 1:15,000 dilution was incubated for 45 min at room temperature. Bound antibody was detected using enhanced chemiluminescence (ECL, GE Healthcare) according to the manufacturer’s instructions. The blot shown is representative of two independent biological replicates. Microscope images were acquired using an Axio Imager 1 (Zeiss).