Immunofluorescent Visualization of Ciliary Structures

To visualize cilial structures, immunofluorescence was performed using an anti-acetylatedα-tubulin antibody (1:1000, T6793, Sigma) and an anti-γ-tubulin antibody (1:1000, T3320, Sigma) [67 (link)]. Briefly, cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature. Fixed cells were permeabilized with 0.05% Triton X-100 and then incubated with the primary antibodies overnight at 4° C. Alexa Fluor 568-conjugated anti-rabbit (1:1000, A-11011, Invitrogen) or Alexa Fluor 647-conjugated anti-mouse (1:1000, A-21235, Invitrogen) antibodies were used as secondary antibody. Counter staining of nuclei was done with DAPI (Sigma) [68 (link)]. The experiments were conducted in quadruplicate. The same treatment was used for Ki67 (1:1000, J3009, Santa Cruz Biotechnology, USA), PCNA (1:1000, A5324, Selleckchem, USA), β-catenin (1:200, A5038, Bimake, USA) staining and Wnt3a (1:200, 2721, Cell Signaling Technology, USA) staining as well as for Osteocalcin (OCN) (1:1000, 16157-1-AP, Proteintech, USA) staining. Ki67 was visualized using a FITC-conjugated goat anti-rabbit (1:1000, 0110119-0100, BBI) antibody.