Immunohistochemical (IHC) and immunofluorescent staining

Bromodeoxyuridine (BrdU) (Roche Diagnostics) was used for BrdU cell proliferating assay. BrdU in PBS was injected intraperitoneally to the pregnant mice at a dose of 100 mg BrdU/kg body weight one hour before dissection. E11.5 fetal heads were collected, fixed with 4% PFA, and prepared to be embedded in the O.C.T. compound. Other immunohistochemical analyses were performed on untreated fixed samples. Samples were cut at 12 μm. Sections were incubated with mouse anti-BrdU (1:200, 11170376001, Roche Diagnostics), rabbit anti-cleaved caspase3 (1:200, #9661, Cell Signaling Technology), or anti-Acetylated Tubulin (1:200, T7451, Sigma-Aldrich), double immunohistochemical staining with mixtures of rat anti-PDGFRα (1:500, 14-1401, eBioscience) and rabbit anti-SOX9 (1:1500, cat.#AB5535, Millipore), mouse anti- β-catenin (1:500, 610153, BD BioSciences) and rabbit anti-RUNX2 (1:150, #12556, Cell Signaling Technology). SOX9 and RUNX2 fluorescence signals were detected by anti-Rabbit Alexa Fluor 555 (1:300, A31572, Molecular Probes). Other stainings were processed with biotinylated anti-mouse IgG (1:200, ZA0409, Vector Laboratories), biotinylated anti-rabbit IgG (1:200, ZB1007, Vector Laboratories), biotinylated anti-rat IgG (1:200, BA-4001, Vector Laboratories) followed by Avidin–biotin complex (Vectastain) and 3,3′-diaminobenzidine (Sigma-Aldrich), or Alexa Fluor 488 streptavidin conjugate (1:300, {"type":"entrez-protein","attrs":{"text":"S11223","term_id":"112468","term_text":"pir||S11223"}}S11223, Molecular Probes). IHC experiments were conducted at least three times on different samples.