Anti dlx2

The condyles of the P90 control and Wnt1Cre::iZEG-Dlx2 mice were demineralized and embedded in paraffin, and sectioned at a thickness of 5 µm. The immunohistochemistry process was performed as previously described (11 (link),16 (link)). Briefly, antigen retrieval for slides was performed with a bone antigen restoration liquid kit (Sunteam Biotech Co., Ltd., China) and subsequently, the samples were blocked for 60 min with 3% bovine serum albumin in phosphate-buffered saline (PBS) containing 0.2% Triton-X-100. Subsequently, the sections were incubated with anti-Dlx2 (1:100; Abcam, Cambridge, UK; cat. no. ab18188), anti-osteocalcin (OCN; 1:200; Abcam; cat. no. ab93876) or anti-msh homeobox 2 (Msx2; 1:100; Abcam; cat. no. ab69058) overnight at 4°C. Following incubation, donkey anti-rabbit secondary antibodies (AlexaFluor 488-conjugated; Thermo Fisher Scientific, Inc.; cat. no. A21206) were diluted in PBS (1:300) and incubated for 60 min at room temperature. Finally, the slides were mounted with Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole (Invitrogen; Thermo Fisher Scientific, Inc.) was used to mount the cover-slips. Images were capture under a fluorescence microscope.