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).

Immunohistochemical (IHC) staining was done using HRP‐DAB Cell & Tissue Staining Kit (R&D Systems) according to the manufacturer's protocol. Briefly, paraformaldehyde‐fixed and paraffin‐embedded tissue samples were cut into 5 μm‐thick sections. Sections were deparaffinized, heat retrieved, blocked and thereafter incubated with the primary antibodies (DSPP, sc‐73632, 1:200, Santa Cruz Biotechnology) overnight at 4°C, followed by washing and incubation with HRP‐conjugated secondary antibodies. Signals were developed with DAB finally. The experiments were repeated at least 3 times.