Ab32570

Shaved dorsal skin was mounted on 3 mm blotting paper and placed in 4% methanol-free formaldehyde (Thermo Fisher Scientific, 28906) at 4°C for 1 hour. The skin was then washed with PBS and transferred to 18% sucrose overnight for cryoprotection before embedding in OCT embedding matrix (Cellpath, KMA-0100-00A) and frozen on dry ice. Then 7 µm cryotome sections were dried at room temperature in the dark for 30 min and washed in PBS containing 0.05% Tween 20 (Sigma-Aldrich, P2287). To image endogenous tdTomato fluorescence, sections were incubated with 1 µM DAPI for 10 min, washed twice with PBS and mounted with Prolong Gold (Thermo Fisher Scientific, P36930). For indirect immunofluorescence staining, sections were incubated with blocking buffer [PBS containing 5% goat serum (Vector, S-1000) and 0.3% Triton X-100 (Sigma-Aldrich, T8787)] for 30 min, then incubated with primary antibodies PDGFR-β (1:25, Abcam, Ab32570) or CD31 (1:50, BD Pharmingen, 550274) for 2 h. Sections were then washed twice and incubated for 30 min with Alexa fluor 488 conjugated secondary antibodies [Molecular Probes; goat anti-rabbit 488 (1:1000, A11034) or goat anti-rat 488 (1:1000, A11006)], washed twice, incubated with DAPI and mounted as described above. Images were obtained using a Zeiss LSM780 confocal microscope.

Femurs from the above fractured mice or Ng2-CreER;Rosa26R^tdTomato mice were fixed in a 4% paraformaldehyde solution for 1 h and then incubated in a 25% sucrose solution overnight. Then, the samples were embedded in 4% carboxymethyl cellulose (CMC, Leica Microsystems, Tokyo, Japan) and frozen in liquid nitrogen. The embedded samples were cut into 7 µm sections by a cryostat (CryoStar NX70, Thermo Fisher Scientific Inc.). The distribution of GFP or tdTomato-expressing cells in the newly developed callus was investigated with a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan). Immunohistochemistry analyses were also performed on frozen sections as previously described [43 (link)]. Antibodies against NG2 (Miltenyi Biotec, #130-097-455), PDGFRβ (Abcam, Cambridge, UK, #ab32570), CD31 (BD Pharmingen, #550274), ALP (kindly gifted by Dr. Kimimitsu Oda, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan), Osterix (Santa Cruz Biotechnology, Inc., Dallas, TX, USA, #sc-22536-R), Sost (R&D systems, #AF1589), Runx2 (R&D systems, #MAB2006), and type 1 collagen (Abcam, #ab34710) were used as primary antibodies. Stained sections were observed using a fluorescence microscope or a confocal microscope (A1R; Nikon, Tokyo, Japan).