Axioskop a1 microscope

Histological images were collected on a Zeiss Axioskop A1 microscope using a MrC3 camera and the Zen imaging software. Flourescent immunoflourescence images were captured on an Olympus Flou-View 100 confocal microscope. The images were compiled in Adobe Photoshop. Wholemount images were captured on a Leica M165FC dissecting microscope using a DFC450 camera with the Leica LAS software. We measured the proximal-distal length of the nasal, frontal and parietal bone as well as the total length of the skull vault using Leica Application Suite software (LAS v4.4). We used GraphPad Prism 5 to perform the Student’s t-test (p < 0.05).
The datasets generated during and/or analyzed during the current study are available from the corresponding author on request.

C57BL/6J WT and Trps1^−/− mice were fixed in either 4% paraformaldehyde (for detection of Trps1 and Tgfβ3) or Carnoy’s solution (for detection of chondroitin sulfate proteoglycan (CSPG), β-catenin, and Twist1) and embedded in paraffin. Heat-induced epitope retrieval was performed in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween20, pH = 6.5). The primary antibodies used were 1:50 rabbit anti-TRPS1 (Abnova, PAB17465), 1:200 anti-chondroitin sulfate proteoglycan (Sigma, C8035), 1:100 rabbit anti-TGFβ3 (Abcam, ab15537), 1:250 rabbit anti-β-catenin (Abcam, ab50581), 1:100 rabbit anti-Twist1 (Abcam, ab32572), and 1:20 anti-Ki67 (Santa Cruz, sc-7846). Anti-rabbit AlexaFlour 488-conjugated secondary antibody (Thermo Fisher Scientific) and ProLong Gold with DAPI Antifade Mountant (Molecular Probes, Thermo Fisher Scientific) were used. Images were taken with Zeiss AxioCam on a Zeiss Axioskop A1 microscope and ZEN software.