Ab9572

Bone samples harvested from patients, and implants from mice, were fixed and decalcified in 10% ethylenediaminetetraacetic acid at pH 7.4 for 1 month. The maximum cross-sectional area of the calluses was selected as the embedding surface, which was parallel to the bottom of the embedding box. Thus, the maximum cross-sectional area of the calluses could be included in the sections. To ensure that the mid part of the regenerating callus was included in histological sections, rough trimming with sectioning was performed until the maximum cross-sectional area of callus tissue was nearly exposed in the sections, and serial sectioning (5 μm) was then started. In total, 60 sections per sample were collected. The sections were stained with H&E, Gomori, immunohistochemistry, and Masson’s trichrome. The number of osteoblasts was quantitated in five nonconsecutive sections per sample after H&E staining. Values for the BV/TV, Tb.Th, Tb.N, and Tb.Sp were calculated by H&E staining, as described by Zhang et al.36 (link). In addition, the area of new bone formation induced by callus in nude mice was measured in sections using IPP software. Immunohistochemistry was performed with a mouse anti-BMP2 monoclonal antibody (#ab6285; Abcam, Cambridge, UK), and rabbit polyclonal antibodies against FGF2 (#ab16828), TGFB1 (#ab66043), and IGF1 (#ab9572).

Protein lysates were generated using the mammalian protein extraction reagent RIPA (Beyotime, Shanghai, China). The concentration of extracted total protein from each sample was calculated using the BCA Protein Assay Kit (Thermo Pierce, Rockford, IL, USA). The equivalent protein for each sample was loaded into a 10% sodium docedyl sulfate-polyacrylamide gel electrophoresis and fractionated, and the denatured proteins were subsequently transferred from gel to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA) by a Mini-PROTEAN Tube Cell instrument (Bio-Rad, Hercules, CA, USA). The membranes were incubated with antibodies (IGF-1, ab9572, Abcam; glyceraldehyde 3-phosphate dehydrogenase, ab8245, Abcam) overnight at 4°C and then with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody for 1 h at room temperature. The enhanced chemiluminescence substrate (Beyotime) was used to visualize the band, and a picture was captured by an imaging system (UVP, Upland, CA, USA). Finally, the quantification analysis was performed by ImageJ 1.45 software (NIH Image).

Cells were rinsed in PBS, fixed with 4% PFA/PBS for 10 min, and permeabilized with 0.25% Triton X-100/PBS for 10 min at room temperature. Samples were blocked with 3.5% BSA/PBS 1 h at room temperature and incubated with primary antibodies overnight at 4 °C. Cells were incubated with appropriate fluorescent secondary antibodies (1:250; Invitrogen) 1 h at room temperature and mounted with hard-setting antifade mounting agent (Aqua-Tex). All antibodies were diluted in 3.5% BSA/PBS. Primary antibodies used were anti-MAP2 (1:2,000, M4403; Sigma), anti-D2R (1:500, AB5084P; Chemicon), anti-IGF-1 (1:200, ab9572; Abcam), and anti-Fos (1:4,000, ABE457; Millipore).

Proteins were harvested from human NP tissues or mouse disc tissues with a Protein Extraction Kit (Thermo, Massachusetts, USA). Immunoblotting was performed as in a previous study (Miao et al., 2008 (link)), using primary antibodies, against collagen I/X (ab34710/ab58632, Abcam, Cambridge, UK), collagen II (ab34712, Abcam, Cambridge, UK), Sirt1 (ab110304, Abcam, Cambridge, UK), superoxide dismutase 1/2 (SOD1/2) (ab13498/ab13533, Abcam, Cambridge, UK), matrix metalloproteinases-13 (MMP-13) (ab52915/ab39012, Abcam, Cambridge, UK), nuclear factor kappa-B-p65 (NF-κB-p65) (SC-71675, Santa Cruz, California, USA), insulin-like growth factor 1 (IGF-1) (ab9572, Abcam, Cambridge, UK), vascular endothelial growth factor (VEGF) (ab69479, Abcam, Cambridge, UK), p19/53 (SC-1665/SC-126, Santa Cruz, California, USA), cyclin-dependent kinases 4/6 (CDK4/6) (ab199728/ab131469, Abcam, Cambridge, UK), retinoblastoma protein/phosphorylated retinoblastoma protein (Rb/pRB) (SC-74562/SC-56175, Santa Cruz, California, USA), transcription factor E2F1/2 (E2F1/2) (SC-137059/SC-633, Santa Cruz, California, USA), and β‐actin (ab8226, Abcam, Cambridge, UK). Immunoreactive bands were analyzed by Scion Image Beta 4.02 and visualized with ECL (Beyotime, Shanghai, China).

Used for immunohistochemistry, the primary antibodies included rabbit anti‐EGFR antibody (1:200 dilution, Ab52894, Abcam, Cambridge, MA, USA), rabbit anti‐IGF1antibody (1:200 dilution, Ab9572, Abcam, Cambridge, MA, USA), rabbit anti‐Vim polyclonal antibody (1:200 dilution, 10366‐1‐AP, Abcam, Proteintech Wuhan, China). Meanwhile, Alexa Fluor 488‐, 594‐, 647‐conjugated secondary antibodies were used for detection with the primary antibodies (1:200 dilution, Jackson ImmunoResearch). EGFR Monoclonal Antibody, APC (Sc‐373746, SantsCruz Biotechnology, CA, USA), CD45 Monoclonal Antibody, FITC (157214, Biolegend, CA, USA), CD31 Monoclonal Antibody, FITC (102405, Biolegend, CA, USA) were used for EGFR⁺ cells sorting according to manufacturer direction.