Ab150077

To examine extracellular matrix formation, three paraffin sections of each tooth (n = 4) on day 28 were immunostained using rabbit anti-periostin (ab92460, 1:500; abcam, Cambridge, UK) and goat anti-rabbit Alexa 488-conjugated secondary antibody (ab150077, 1:200; abcam) followed by counterstaining with Hoechst 33342. The positive area relative to the regenerated area was analyzed using a BZ9000 BIOREVO fluorescence microscope. The ratio of periostin-positive cells to Hoechst 33342-positive cells in the regenerated tissue on day 28 was calculated in three sections of each tooth (n = 4).
Furthermore, immunohistological analysis was performed using a rabbit anti-asporin/periodontal ligament-associated protein 1 (PLAP-1) (ab58741, 1:500; abcam) antibody with a goat anti-rabbit Alexa 488-conjugated secondary antibody (ab150077, 1:200; abcam) followed by counterstaining with Hoechst 33342. The positive area relative to the regenerated area was analyzed using a BZ9000 BIOREVO fluorescence microscope. The ratio of PLAP-1-positive cells to Hoechst 33342-positive cells in the regenerated tissue on day 28 was calculated in three sections of each tooth (n = 4).

MLO‐Y4 cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X‐100 for 5 min. Then, the cells were incubated with a rat anti‐mouse primary antibody against sclerostin (Abcam, 86465) mouse anti‐mouse primary antibody against β‐catenin (Proteintech, 66379) and rabbit anti‐mouse primary antibody against Cx43 (Abcam, ab11370) for 12 h at 4°C. Following three washes in PBST, the cells were incubated with goat anti‐rat Alexa Fluor®‐594 (Abcam, ab150160) secondary antibody, goat anti‐mouse Alexa Fluor®647 (Abcam, ab150115) secondary antibody and goat anti‐rabbit Alexa Fluor®488 (Abcam, ab150077) secondary antibody for 1 h in a dark room. Then cells were washed in PBST and nuclei were stained with DAPI for 5 min. These stained cells were observed under a laser‐scanning confocal microscope LSM880 (Zeiss) with an excitation wavelength of 594, 647 and 488 nm.
To detect cleaved‐caspase‐3, MLO‐Y4 cells were incubated with rabbit anti‐mouse primary antibody against cleaved‐caspase‐3 (CST, 9664T) and then incubated with goat anti‐rabbit Alexa Fluor®488 (Abcam, ab150077) secondary antibody. MLO‐Y4 cells were observed with an excitation wavelength of 488 nm.

Total proteins were extracted from tissues or fibroblasts using a Total Protein Extraction Kit (Sangon) according to the manufacturer’s instructions, and then the protein concentration was measured with an Enhanced BCA Protein Assay Kit (Beyotime). After the samples were denatured at 100°C for 10 min, equal amounts of protein extract were subjected to electrophoresis in 10% SDS-PAGE gels and then transferred to PVDF membranes (Biosharp). The membranes were blocked with 5% nonfat milk in PBS at room temperature for 1 h and then incubated overnight at 4°C with primary antibodies. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h, and signals were detected using ECL western blotting Substrate (Tanon). The antibodies used were as follows: anti-uPA (PA5-34638, Invitrogen), anti-uPAR (ab103791, ab221680, Abcam), anti-PAI-1 (ab66705, Abcam), anti-Collagen I (ab260043, Abcam; bs-10423, Bioss), anti-α-SMA (ab124964, Abcam; 19245, Cell Signaling), anti-GAPDH (ab181602, Abcam), anti-SMAD1+SMAD5 polyclonal(bs-2973R, Bioss), anti-rabbit IgG (ab150077, Abcam), anti-phospho-SMAD1+SMAD5(Ser463+Ser465) polyclonal antibody (bs-3418R, Bioss), Anti-MADH7/SMAD7(Abcam, ab216428), Anti-PPAR Gamma (ser112) Polyclonal Antibody (Bioss, bs-3737R), anti-rabbit IgG (ab150077, Abcam), and anti-rabbit IgG (8889, Cell Signaling).