High resolution fluorescence microscope

OCCM-30 cells were cultured overnight on sterile Falcon™ Chambered Cell Culture Slides (354108, Fisher Scientific) and further fixed with 4% paraformaldehyde (30525-89-4, Sigma-Aldrich) for 10 min at room temperature. Cells were permeabilized with 0.5% Triton™ X-100 Surfact-Amps™ Detergent Solution (28313, Thermo-Fisher) for 20 min. Then, cells were incubated in blocking buffer containing 10% goat serum, 0.3 M glycine, 1% BSA (071M8410, Sigma) and 0.1% Tween-20 (P1379, Sigma-Aldrich) for 30 min at room temperature and further incubated with primary antibodies AdipoR1 (ab70362, Abcam) (dilution 1:250) or AdipoR2 (ab77612, Abcam) (dilution 1:250) at 4°C overnight. The secondary antibodies DyLight 488 polyclonal goat anti-rabbit (ab96899, Abcam) (dilution 1:500) or donkey anti-goat Alexa Fluor 647 (ab150131, Abcam) (dilution 1:500) conjugated to fluorescein isothiocyanate were used. After washing with 1× phosphate-buffered saline (PBS) (10010023, Thermo-Fisher), samples were mounted using a fluorescent Mounting Medium with DAPI (ab104139, Abcam). Staining was analyzed using a high-resolution fluorescence microscope (Leica Microsystems, Wetzlar, Germany) and photographed.

Cementoblasts were cultured on sterile Falcon^TM Chambered Cell Culture Slides (354108, Fisher Scientific) until 50% confluence and afterward were fixed with 4% paraformaldehyde (Cat: 158127, Sigma-Aldrich) dissolved in 1X phosphate-buffered saline (PBS) (1401683, Gibco), adjusted to pH 7.4, for 10 min at room temperature, and permeabilized with 0.5% Triton^TM X-100 Surfact-Amps^TM Detergent Solution (28313, Thermo-Fisher) for 20 min. Then, cells were kept in a blocking buffer containing 10% goat serum, 0.3 M glycine, 1% BSA (071M8410, Sigma-Aldrich), and 0.1% Tween-20 (P1319, Sigma-Aldrich) for 30 min at room temperature and incubated with primary antibodies for GSK-3β (1:500, #12456, Cell Signaling Technology) and β-catenin (1:500, #8480, Cell Signaling Technology) at 4°C overnight. After washing three times with 1X PBS (1401683, Gibco)–0.1% TRITON X-100 (T-9284, Sigma-Aldrich) for 5 min, the cells were incubated with DyLight 488 goat anti-rabbit polyclonal secondary antibody (1:1,000, ab96899, Abcam), which conjugated to fluorescein isothiocyanate for 1 h. After washing with 1X PBS-Tween-20, DNA was stained using a fluorescent mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (ab104139, Abcam) for 15 min. Staining was analyzed using a high-resolution fluorescence microscope (Leica Microsystems, Wetzlar, Germany) and photographed.