Af2909

Cranial base bones were fixed in 4% formalin solution for 24 hours, decalcified in 10% EDTA solution for 24 hours and paraffin‐embedded. Five‐micron sections were cut horizontally and stained with Alcian Blue (Sigma) for 30 minutes and counterstained with Nuclear Fast Red (Millipore).
For immunofluorescence analysis, sections were blocked 1 hour with 1.5% calf serum, incubated over night at 4°C with anti‐collagen X (Abcam, ab58632), anti‐heparan sulfate proteoglycan (Abcam, ab2501) or anti‐bone alkaline phosphatase (R&D, AF2909) primary antibodies and 1 hour at room temperature with appropriate Alexa Fluor secondary antibodies (1/1000 dilution) (Life Technologies, A11037). Nuclear staining was performed with Dapi (Millipore) and sections were imaged on an Axioplan2 upright microscope (Carl Zeiss Microscopy GmbH) with Zeiss 4X, 10X or 20X Achroplan dry NA 0.1.

After the mice were perfused with PBS and formalin, brains were collected and separated sagittally. One hemisphere was used to dissect the cortex and hippocampus, which was rapidly frozen and stored at −80 °C. Another hemisphere was fixed in 10% buffered formalin phosphate (Cat. SF100‐4 fisher scientific) for 24 h at 4 °C and transferred to 30% sucrose in PBS for another 48 h at 4 °C. Brain tissue was embedded in OCT and cryosectioned coronally at 50 µm with a microtome (Cat. Microm HM 525, Thermo Fisher). For immunofluorescence staining, tissue sections were incubated with specific primary antibodies to mouse PDGFRβ (1:100, ab32570, Abcam), CD13 (1:100, MCA2183, BIO‐RAD), CD31 (1:50, AF3628 R&D), Caveolin‐1 (1:100, 3267S, Cell signaling Technology), ALPL (1:20, AF2909, R&D), MMP14 (1;100, ab51074, Abcam), Lectin (1:200, DL‐1174, Vector), TFRC (1:100, NB100‐64979, Novus) and Albumin (ab106582, 1:100, Abcam) for 24 h at 4 °C followed by corresponding fluorescence‐linked secondary antibodies (Jackson ImmunoResearch Laboratories) for 1 to 4 h while avoiding light. The sections were then mounted with DAPI (8961S, Cell signaling Technology). The sample images were captured by a confocal microscope (Zeiss LSM 780).

Brain and bone sections were prepared as described previously [6 (link), 7 ]. Briefly, after perfusion with PBS and formalin, the brains and bones were collected and separated sagittally. For brain preparation, the brain was fixed in 4% paraformaldehyde solution for 24 h at 4℃, transferred to 30% sucrose in PBS for another 48 h at 4℃, and embedded in OCT and cryosectioned coronally at 20 μm with a cryostat (CM1950, Leica). For bone preparation, the bone was fixed in 4% paraformaldehyde solution for 24 h at 4℃, transferred to 30% sucrose and 2% polyvinyl pyrrolidone in PBS for another 48 h at 4℃, and embedded in OCT and cryosectioned coronally at 10 μm with a microtome. All sections were stored at -80℃ before further staining.
For immunofluorescence staining, tissue sections were incubated with specific primary antibodies to mouse ALPL (1:20, AF2909, R&D), IBA1 (1:200, ab178846, Abcam), GFAP (1:500, ab7260, Abcam), SOX2 (1:100, ab92494, Abcam), ZO-1 (1:100, ab221547, Abcam), Rank (1:100, MA5-16153, Thermo Fisher), PDGF-B (1:100, ab23914, Abcam) for 24 h at 4℃ followed by corresponding fluorescence-linked secondary antibodies (Jackson ImmunoResearch Laboratories) for 1 h while avoiding light. The sections were mounted with DAPI (S2110, Solarbio). Sample images were captured using a confocal microscope (Zeiss LSM 980).