Ab150687

Cell cultures were fixed in cold methanol for 15–20 min. After rinsing, the fixed cells were incubated with (5%) Silver Nitrate Solution (abcam product # ab150687) and crosslinked under UV light for 20–30 min, then rinsed twice with distilled water. The dishes were incubated with (5%) Sodium Thiosulfate Solution for 2–3 min, rinsed twice with distilled water; then finally incubated with Nuclear Fast Red Solution for 5 min, and rinsed several times with distilled water to remove excess stain.

The calcein labeling in methyl acrylate (Polysciences Inc., Warrington, PA, USA)-embedded tibia sections was evaluated using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Trabecular bone histology was evaluated using von Kossa staining kits (ab150687; Abcam, Cambridge, UK) and hematoxylin–eosin stain kits (Sigma-Aldrich, St Louis, MO, USA). Osteoclasts were probed using a tartrate-resistant acid phosphatase (TRAP) staining kit (B-Bridge International Inc., Mountain View, CA, USA). Trabecular volume (BV/TV, %), mineral acquisition rate (MAR, μm/day), and osteoclast number (Oc.N/mm) were quantified using Axio Image Analysis System (Carl Zeiss, Oberkochen, Germany). Six random fields in each section and 3 sections in each animal were selected for histomorphometry. Immunohistochemical staining for paraffine-embedded sections was performed using β-galactosidase (ab136776, Abcam, Cambridge, UK), p16^Ink4a (1E12E10, Invitrogen Thermo Fisher Scientific Inc., Waltham, MA, USA), 5-methylcytosine (5mC) (GT411, Invitrogen, Carlsbad, CA, USA), and 8-hydroxydeoxyguanosine (8-OHdG) (BS-1278R; Thermo Fisher Scientific Inc., Waltham, MA, USA). Immunostained osteoblasts in each high-power field (×400 magnification) were counted. Three random fields in each section and 3 sections in each tibia specimen were selected to quantify immunostained cells.

After overnight fixation in 10% neutral formalin, the aortic samples were embedded in OCT frozen section compound (Leica REF 3801480, Wetzlar, Hesse, Germany), and cut into 10–12 µm sections for hematoxylin and eosin (H&E), Oil-red O, and Masson’s trichrome staining as described previously (Tung et al., 2020 (link)). H&E staining was performed to examine the sizes of atherosclerotic plaques, Oil-red O staining was performed to assess lipid deposition, and Masson’s trichrome staining was performed to identify the collagen fiber contents in the tunica intima of the aortic root. Quantification was performed by inspecting 4 (slides 3–6) of 9 serial sections of the aortic root tissues from each mouse, as described previously (Tung et al., 2020 (link)). The deposition of calcium minerals in atherosclerotic lesions of the aortic roots was identified using a von Kossa stain kit (ab150687; Abcam).

von Kossa staining was applied to detect phosphate ions (PO₄³⁻). Following the staining protocol (Abcam, ab150687), the sections were dewaxed, rehydrated, and incubated with 5% silver nitrate for 45 min under ultraviolet rays. After rinsing several times with fresh distilled water, the sections were incubated with 5% sodium thiosulfate solution for 10 min to wash the unreacted silver ions, followed by a nuclear fast red solution.