Materials and methods
Animals
Japanese Black Cattle were raised in cattle farms for 30 months and slaughtered at the slaughterhouse for meat products (Kobe Chuo Chikusan, Kobe, Japan). Bovine legs were chosen by staff at the slaughterhouse, not by researchers. Explant culture started within 24 h after the animal’s death. This study used dead bovine legs, and no treatment or experimental intervention for living animals was performed.
Preparation of periosteal samples
The periosteum was separated from the bovine leg as described previously [7]. The observation period was from day 0 to 7 weeks. After 2 weeks of culture, without periosteum-derived cells, cell culture dishes and the periosteum were excluded. Explants that had contamination at any period were excluded. Legs from six different cows were used, and in some cases, the same cow at different time points was used. Bone sections were removed, fixed with 4% paraformaldehyde, and cast into paraffin blocks. The periosteum was cultured in 100-mm dishes in Medium 199 supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and 5 mg/mL ascorbic acid for up to 7 weeks. The medium was changed once a week. Every week, the periosteum was fixed and sections were prepared. Additional file 1: Fig. S1a, b shows the schema of this study. After the periosteum was peeled, cortical bone was excised and fixed. Before explant culture (day 0), the periosteum and bone with the cambium layer were investigated (Additional file 1: Fig. S1a). After explant culture, the periosteum and periosteum-derived cells were investigated at weeks 1 to 7 (Additional file 1: Fig. S1b).
Fluorescent immunostaining and immunohistochemistry
All paraffin sections were pretreated with ready-to-use Proteinase K (Dako Cytomation, Glostrup, Denmark) for 10 min for antigen retrieval. The primary antibodies used were mouse anti-bovine osteocalcin monoclonal antibody (code no. M042, clone no. OCG2; Takara Bio Inc., Shiga, Japan) and goat anti-FBXW2 polyclonal antibody (#PA5-18,189; Invitrogen, Eugene, OR, USA). The secondary antibodies used were goat anti-mouse Alexa Fluor™ 488-labeled antibody (#A11029; Invitrogen), mouse anti-goat IgG-CFL 594 (no. sc516243; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and N-Histofine Simple Stain AP (multi; #414,261; Nichirei Biosciences Inc., Tokyo, Japan). The alkaline phosphatase-tagged antibodies were visualized with PermaRed/AP (K049; Diagnostic BioSystems, Pleasanton, CA, USA). Incubation with the anti-bovine osteocalcin monoclonal antibody (diluted 1:500) and anti-FBXW2 antibody (diluted 1:100) was performed for 4 h at room temperature. Cell nuclei were stained with hematoxylin or 4′,6-diamidino-2-phenylindole (DAPI). For the negative controls, an antibody against the receptor activator of NF-κB ligand (RANKL; No. sc-377079; Santa Cruz Biotechnology, Inc.) and normal goat serum (143–06,561; Fujifilm Wako Pure Chemical Industries, Ltd., Kanagawa, Japan) were used. Images were obtained using a fluorescence microscope (BZ-9000; Keyence Japan, Osaka, Japan) and BZ-II Viewer (Version1.1; Keyence) and BZ-II Analyzer (Version1.1; Keyence) software. One researcher performed all stages of the experiments and data analysis.