Anti dspp sc 73632

For immunofluorescence staining, cultured cells and organoids were fixed for 20 min, permeabilized with 0.1% Triton X-100 for 15 min, washed thrice with PBS for 5 min, and blocked with 10% bovine serum albumin for 1 h. Cells were incubated with the primary antibody anti-SCUBE3 (ab189955; Abcam, Cambridge, UK), and the organoids were incubated with anti-DSPP (sc-73632; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight. Sections of tooth root fragments were stained with DSPP (sc-73632; Santa Cruz) and α-tubulin (sc-8035; Santa Cruz). After washing thrice with PBS for 5 min, cells and organoids were, respectively, incubated with Dylight 594 (35560; Thermo Fisher Scientific, Waltham, UK) and CoraLite 488-conjugated (CL488-66122; ProteinTech Group, Chicago, IL, USA) secondary antibodies for 1 h in the dark. DAPI (Sigma-Aldrich) was used to stain the nuclei. Finally, the cells were observed under a Leica DMI4000 B fluorescence microscope (Leica Imaging Systems, Cambridge, UK). Organoids were transferred to observation dishes specified for confocal laser microscopy and examined with a confocal microscope (Carl Zeiss, Göttingen, Germany).

Total cell proteins were lysed using radioimmunoprecipitation assay (Beyotime) according to the manufacturer’s protocol. Proteins in the conditioned medium were extracted using a liquid sample total protein extraction kit (Solarbio, Beijing, China). Western blotting was performed as previously described [10 (link)]. The primary antibodies used were anti-SCUBE3 (ab189955; Abcam), anti-AMBN (orb155652; Biorbyt, Cambridge, UK), TGFβR1 (ab31013; Abcam), anti-DSPP (sc-73632; Santa Cruz), anti-DMP1 (ab103203; Abcam), anti-OPN (ab8448; Abcam), anti-OSX (ab13418; Abcam), anti-BMP2 (ab14933; Abcam), anti-BMPR1A (ab264043; Abcam), anti-p-SMAD1/5 (9516; Cell Signalling Technology, Boston, MA, USA), and anti-SMAD1 (D59D7; Cell Signalling Technology). Anti-GAPDH (Rayantibody, Beijing, China) was used as an internal control. Antibody binding was detected using an enhanced chemiluminescence kit (Millipore, Billerica, MA, USA). The intensity of the bands was quantified using the ImageJ software (NIH, USA).