Stereo discovery v20 stereomicroscope

Tartrate resistant acid phosphatase (TRAP) staining was performed using a commercially available acid phosphatase kit from Sigma-Aldrich (Cat No. 387, Sigma Aldrich, Schnelldorf, Germany). The staining was performed according to the manufacturer's instructions. In detail, after aspiration of the culture medium, the cells were fixed with 100 μL fixative solution (3.316 mL acetone, 1.276 mL citrate solution (Sigma Aldrich, Schnelldorf, Germany), and 0.408 mL 37% formaldehyde) for 30 s. Thereafter the samples were washed twice with 200 μL PBS. Then, 100 μL of a freshly prepared diazotized Fast Garnet GBC Base Naphthol AS-BI phosphoric acid staining solution (Sigma Aldrich, Schnelldorf, Germany) was added to the wells followed by incubation at 37 °C and 5% CO₂. After 60 min the staining solution was aspirated, the surfaces were washed twice with 200 μL dH₂O and dried overnight at RT. The transmitted light microscope VisiScope IT 404 (VWR International, Leuven, Belgium) and the software ZEN 2.3 (blue edition) from Carl Zeiss Microscopy GmbH (Zeiss, Oberkochen, Germany) were used to analyze the PS reference. The scaffolds were analyzed with the SteREO Discovery.V20 stereomicroscope and the ZEN 2012 (blue edition) software (both Zeiss GmbH, Oberkochen, Germany).

Whole-mount ISH was carried out as described previously (Kee and Bronner-Fraser, 2001 (link)). Digoxigenin-labelled probes were synthesized from linearized vectors containing partial full-length cDNAs of FolR1, Rfc1, Dnmt3a, Dnmt3b, Tet1, Tet2, Tet3, Bmp4m and Notch1. Hybridized probes were detected using an alkaline phosphatase-conjugated antidigoxigenin antibody (Roche, 1:200) in the presence of NBT/BCIP substrate (Roche). Embryos were photographed as a whole-mount using a ZEISS SteREO Discovery V20 Stereomicroscope (Axiocam 512 color) and Carl ZEISS ZEN2 (blue edition) software.

For SEM investigation, the adhesive cements were bonded to hydroxyapatite cement test specimens (made from α-TCP and 2.5% Na₂HPO₄) and these were then shear tested. Furthermore, the dried samples were coated with platinum at a thickness of 4.0 nm in the EM ACE600 sputter coater (Fa. Leica Camera, Wetzlar, Germany) at 10⁻⁷ mbar to improve SEM resolution. Scanning electron micrographs were taken on the Zeiss Crossbeam 340 scanning electron microscope (Carl Zeiss, Oberkochen, Germany) at 3.00 kV, a vacuum of 1.25 × 10⁻⁶ mbar, and an aperture of 30.00 µm. Microscopic images of the adhesive residues on the bone were also taken with the SteREO Discovery.V20 stereomicroscope (Carl Zeiss Microscopy GmbH, Jena, Germany) using a 81 mm PlanApo 0.63x FWD lens. The ZEN® software (Carl Zeiss Microscopy GmbH, Jena, Germany) was used for this purpose.