Ab85799

AS fibroblasts were lysed with RIPA buffer (Beyotime Shanghai, China) to extract total protein. After quantified using BCA Kit (Beyotime), equal amounts of proteins were separated with SDS-PAGE gel and transferred onto PVDF membranes (Millipore). Then, the membranes were blocked with 5% non-fat milk and incubated with antibodies against runt-related transcription factor 2 (RUNX2; 1:1,000, ab76956, Abcam, Cambridge, MA, United States), osteocalcin (OCN; 1:500, ab93876, Abcam), osteoprotegerin (OPG; 1:1,000, ab73400, Abcam), SOST (1:1,000, ab85799, Abcam) or β-actin (1:5,000, ab8227, Abcam) at 4°C overnight. After incubated with secondary antibodies against Goat Anti-Rabbit IgG (1:10,000, ab205718, Abcam) or Goat Anti-Mouse IgG (1:5,000, ab205719, Abcam), protein signals were visualized using enhanced chemiluminescence (ECL) reagent (Yeasen). The experiment was performed three biological replicates.

Antigen retrieval was carried out by incubating the sections with hyaluronidase (15.000 U/mL, Sigma‐Aldrich) in a hot cabinet at 37°C for 30 minutes, followed by EnVision FLEX Wash Buffer 1× (Dako‐Agilent, Santa Clara, CA, USA). Sections were blocked with endogenous peroxidase (Dako‐Agilent) for 5 minutes and 1% BSA (Sigma‐Aldrich) for 30 minutes, both at room temperature (RT), before incubation with the respective primary antibodies: rabbit dentin matrix acidic phosphoprotein 1 (DMP1) (1:200; HPA037465; Sigma‐Aldrich), rabbit sclerostin (1:50; ab85799; Abcam, Cambridge, UK), sheep Podoplanin (E11) (1:50; AF3670; R&D Systems, Minneapolis, MN, USA), and mouse osteocalcin (OCN) (1:00; 33–5400; Thermo Fisher Scientific) overnight at 4°C. Incubation with appropriate secondary antibodies envision + system‐HRP anti‐rabbit (Dako‐Agilent), anti‐mouse (Dako‐Agilent), and IgG HRP‐conjugated anti‐sheep (1:500; R&D Systems) were performed for 30 minutes at RT followed by washing. Finally, the sections were stained with hematoxylin, dehydrated, and mounted. For the negative control, sections were stained without primary antibodies. The mounted sections were analyzed using an Olympus BX51 microscope.

The slides were incubated with an anti-DKK1 antibody (ab61034, Abcam, Cambridge, UK), anti-SOST antibody (ab85799, Abcam, Cambridge, UK), anti-PSTN antibody (ab92460, Abcam, Cambridge, UK), anti-FGF23 antibody (LS-C293901, LSBio, Seattle, WA, USA), anti-OPG antibody (ABIN668556, Antibodies-online, Aachen, Germany), and an isotype control antibody (ab27478, Abcam, Cambridge, UK) overnight at 4 °C. The dilution used in each case was 1:100 with blocking buffer. In the second step, 100 µL of the enzyme-activated secondary antibody (anti-rabbit polymer) bound to the primary antibody and was visualized with the addition of hydrogen peroxide (H₂O₂) and the chromogen DAB (3.3′-diaminobenzidin). The time of incubation was one to four minutes. After the secondary step, the slides were counterstained with hematoxylin for the visualization of the cell nuclei.