2.8. Immunofluorescence staining

VICs were cultured on the coverslip and treated with and without calcification medium in the absence of phenol red, supplemented with 5 nmol/L AP39 for 5 days. After treatment, the cells were fixed with 3.7% formaldehyde for 15 min and blocked with 10% goat serum for 1 h at room temperature. Mouse anti-human TNF-α (Santa Cruz; sc-52746; 100 μg/mL) and rabbit anti-human IL-1β (Invitrogen; 17h18116; 400 ng/mL) at 1:500 dilution were used as primary antibodies to show levels of TNF-α and IL-1β in VICs. TNF-α antibody was labeled with goat anti-mouse Alexa Fluor 488 fluorophore (Thermo Fisher Scientific; A11004) and IL-1β was labeled with goat anti-rabbit Alexa Fluor 488 fluorophore (Thermo Fisher Scientific; A11070) for 1 h in dark at room temperature. All secondary antibodies were used at 1:500 dilution. Hydroxyapatite crystals were stained with IVI Sense Osteo 680 Fluorescent Probe (OsteoSense; PerkinElmer®; NEV10020EX). Hoechst 33258 was used to stain nuclei. Multicolor STED imaging was acquired with STED (Stimulated Emission Depletion) Leica TCS SP8 gated STED-CW nanoscopy (Leica Microsystem Mannheim, Germany). Gated STED images were deconvolved using Huygens Professional (Scientific Volume Imaging B.V., Hilversum, Netherlands) software. Levels of TNF-α and IL-1β were evaluated by Image J software. Separate images of immunofluorescence stainings (nucleus, osteosense, IL-1β, and TNF-α) with their antibody controls can be found in the Supplementary Fig. 3.