Ab98111

Harvested kidneys were homogenized in RIPA buffer (1% Tween 20, 0.1% SDS, 150 nM NaCl, 10 mM Tris-HCl (pH 7.4), 0.25 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin, 1 mM Na3VO4, 5 mM NaF (Sigma)). Lysates were collected by centrifugation at 15,000 rpm at 4 °C for 10 min. Equivalent amounts of protein were separated by SDS/PAGE and transferred to a PVDF membrane (EMD Millipore Corporation). Proteins were detected using anti-Klotho (ab98111, abcam, Cambridge, UK), anti-NaPi-IIa (ab182099, abcam) and anti-Actin (A2066, Sigma) antibodies. Bands were visualized using ECL Western Blotting Detection Reagent (GE Healthcare, Uppsala, Sweden).

The porcine ovaries were collected and fixed in 4% paraformaldehyde. IHC was performed on 3 μm paraffin-embedded tissue sections from the tissue blocks. Histological architecture of the ovary was assessed using Hematoxylin and Eosin (H&E)-stained sections, through an optical microscope (Olympus, BX41, Japan). To detect Klotho antigen, the tissue sections were stained with a rabbit polyclonal Klotho antibody (ab98111, Abcam), followed by Envision+System-HRP Labelled Polymer anti-Rabbit antibodies (K4003; Dako). Image processing was performed with a Leica DMI 6000B microscope, using a DFC350 camera. Expression levels were classified into three categories: ‘mild’, ‘moderate’, and ‘strong’.

Kidney, aorta and skin from euthanized mice were fixed in 10% phosphate-buffered formalin, and embedded in paraffin. Tissues were sectioned and stained with hematoxylin and eosin (H&E) and using von Kossa methods. Slides were examined by light microscopy (BIOREVO, BZ-9000 (Keyence, Osaka, Japan)) for tissue mineralization. Relative calcification areas in kidney and aorta were calculated using microscopic image analysis software BZ-II analyzer (Keyence). Kidney sections were also stained using anti-p16 INK4A (10883-1-AP, Proteintech, Rosemont, IN, USA) or anti-Klotho (ab98111, abcam) followed by Alexa488-conjugated goat anti-rabbit IgG H&L (Alexa Fluor^® (ab150077, abcam), and nuclei were stained with DAPI (Wako Pure Chemicals Industries, Osaka, Japan). Senescence-associated beta-galactosidase was stained using a SA-β-gal kit (#9860, Cell Signaling, Danvers, MA). Then, sections were observed under a fluorescence microscope (Keyence, Osaka, Japan).