Anti sp7 osterix

Total protein (50 μg) was separated in a Bis-Trispolyacryl amide gel and transferred onto a PVDF membrane (Millipore). The membrane was then incubated in 5% bovine serum albumin (BSA), and then incubated with a primaryantibody at 4°C overnight. Next, samples were incubated with IRDye800CWHRP-conjugated anti-IgG at room temperature for 1hour and visualized via chemiluminescence with an infrared laser scanning system (OdysseyLicor,Lincoln,NE,USA). The following primary rabbit-anti-human antibodies were used: anti-ANGPT2 (1:1000; Abcam); anti-Notch1 (1:500; Abcam); anti-Notch2 (1:500; Abcam); anti-Runx2 (1:1000;Abcam); anti-Sp7/Osterix (1:2000; Abcam); anti-ALP (1:2000; Abcam); anti-OCN (1:500; Abcam) and anti-GAPDH (1:2,500; Abcam).

Femurs from E16.5 C57BL/6 embryos were harvested and fixed in 4% PFA at 4 °C for 24 h. After three washes in PBS, samples were processed for paraffin embedding. Three-micrometer-thick longitudinal cross-sections were obtained using a microtome (RM2255, Leica Biosystems) and stained for toluidine blue. For immunohistochemistry, tissue sections were deparaffinized and rehydrated before heat-induced antigen retrieval (98 °C, 10 mmol·L⁻¹ citrate buffer, pH 6.0). Sections were simultaneously permeabilized and blocked for 1 h at RT in a solution consisting of 0.20% (v/v) Triton X-100, 10% v/v FBS, and 1% v/v BSA in PBS, and incubated with the primary antibody 5E1 (1:100), G3G4 (1:100), and anti-Sp7/Osterix (Abcam, 1:1 000) diluted in blocking solution overnight at 4 °C. Afterward sections were washed and incubated for 1 h at RT with the secondary antibodies (Alexa Fluor 488 and 568, Invitrogen) diluted 1:1 000, in blocking solution. Images were captured on an Axiovert 200 inverted microscope equipped with AxioVision 4.8 software (Zeiss) at the i3S Bioimaging Unit.