mouse monoclonal anti osteocalcin ocn  (Servicebio Inc)

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: CGRP-dependent molecular signaling drives bone marrow stem cell osteogenesis in distraction osteogenesis

doi: 10.3389/fbioe.2025.1641476

Figure Lengend Snippet: CGRP upregulates osteogenic gene and protein expression via the cAMP/PKA/CREB pathway: (A) qRT-PCR analysis of osteogenic transcription factors (Runx2 and Osterix) and marker genes (OPN and OCN), along with cell cycle gene (CyclinD1), after 7 days of osteogenic induction. Data normalized to GAPDH and expressed relative to control. (B, C) Western blot analysis and quantification of osteogenic proteins and CyclinD1 expression. Data represent mean ± SD. *P < 0.05.

Article Snippet: Primary antibody working solutions were then applied, including rabbit polyclonal anti-Runx2 (1:200; Bioss, bs-1134R, Woburn, MA, United States), rabbit polyclonal anti-Osterix (Osx) (1:100; ServiceBio, GB111900 , Wuhan, China), mouse monoclonal anti-osteocalcin (OCN) (1:150; ServiceBio, GB115684 ), rabbit polyclonal anti-osteopontin (OPN) (1:200; ServiceBio, GB11500), rabbit monoclonal anti-phosphorylated PKA (p-PKA Thr197) (1:100; Affinity Biosciences, #AF7246), and rabbit monoclonal anti-phosphorylated CREB (p-CREB Ser133) (1:100; Affinity Biosciences, #AF3189, Cincinnati, OH, United States), with sections placed in humid chambers at 4 °C overnight.

Techniques: Expressing, Quantitative RT-PCR, Marker, Control, Western Blot

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: CGRP-dependent molecular signaling drives bone marrow stem cell osteogenesis in distraction osteogenesis

doi: 10.3389/fbioe.2025.1641476

Figure Lengend Snippet: Immunohistochemical analysis confirms activation of osteogenicmarkers and the signaling pathway: (A) representative immunohistochemical staining and quantitative analysis of osteogenic transcription factors (Runx2 and Osterix), bone matrix proteins (OPN and OCN), and pathway molecules (p-PKA and p-CREB) in distraction gap tissues at 6 weeks of consolidation. Brown staining indicates positive expression. (B) CGRP treatment significantly enhanced all markers (scale bars: 50 μm). Data represent mean ± SD. *P < 0.05.

Article Snippet: Primary antibody working solutions were then applied, including rabbit polyclonal anti-Runx2 (1:200; Bioss, bs-1134R, Woburn, MA, United States), rabbit polyclonal anti-Osterix (Osx) (1:100; ServiceBio, GB111900 , Wuhan, China), mouse monoclonal anti-osteocalcin (OCN) (1:150; ServiceBio, GB115684 ), rabbit polyclonal anti-osteopontin (OPN) (1:200; ServiceBio, GB11500), rabbit monoclonal anti-phosphorylated PKA (p-PKA Thr197) (1:100; Affinity Biosciences, #AF7246), and rabbit monoclonal anti-phosphorylated CREB (p-CREB Ser133) (1:100; Affinity Biosciences, #AF3189, Cincinnati, OH, United States), with sections placed in humid chambers at 4 °C overnight.

Techniques: Immunohistochemical staining, Activation Assay, Staining, Expressing

Journal: Chemical Engineering Journal

Article Title: Extracellular vesicles from mechanical loading stimulated-macrophages favor fracture healing through targeting Adrb2 of osteoblasts

doi: 10.1016/j.cej.2024.159079

Figure Lengend Snippet: Fig. 1. ML stimulates the release of sEVs to promote fracture healing. A) Schematic graph of the ML model and GW4869 injection procedure. 12-week male C57B6/J mice were treated with periodic ML to the distal femur (fractured side) commencing on the 7th day after surgery; the loading treated mice were subsequently administrated with either solvent or GW4869 at the 7th and 10th day following the surgery. B) Representative μCT images of fractured femurs harvested from mice on 14th day post-fracture. C) Quantitative analysis of BV/TV and Tb. N of the callus (n = 5). D, E) Representative images of HE and SO/FG staining in the fractured site. Scale bar: 500 μ m. F, G) Representative images of IF staining and quantitate analysis of RUNX2 and OCN expression in the callus (n = 5). Scale bar: 100 μm. H, I) Representative IF staining and quantitate analysis of the type H vessels areas in the callus (n = 5). Scale bar: 100 μm. J) Flow cytometry plots show the gating strategy for type H vessels in the callus (Live CD45- Ter119- CD31+ EMCN+). K) Quantitative analysis of the number of type H vessels in the callus (n = 5). An individual sample was represented by a dot plot, and data are presented as means ± standard deviation (SD). *p < 0.05, **p < 0.01 and ***p < 0.001, ns: not significant difference. Statistical significance was determined by one-way ANOVA.

Article Snippet: The antibodies were as follows: RUNX2 (Abcam, ab192256; 1:1000), OCN (Takara, M188, 1:150), F4/80 (Invitrogen, 14–4801-82; 1:100), PIEZO1 (Proteintech, 15939–1-AP; 1:400), CD63 (Abcam, ab217345; 1:100), CD31 (R&D Systems, AF3628; 1:100) and Endomucin (Santa Cruz, sc-65495; 1:100), Ki67 (Abcam, ab15580; 1:1000), ADRB2 (Abcam, ab182136; 1:100).

Techniques: Injection, Solvent, Staining, Expressing, Flow Cytometry, Standard Deviation

Journal: Chemical Engineering Journal

Article Title: Extracellular vesicles from mechanical loading stimulated-macrophages favor fracture healing through targeting Adrb2 of osteoblasts

doi: 10.1016/j.cej.2024.159079

Figure Lengend Snippet: Fig. 2. PIEZO1 in macrophages is essential for ML-induced fracture healing. A) Uniform manifold approximation and projection (UMAP) of integrated scRNA-seq data, revealing 11 cell clusters in the callus on 14th day post-fracture. B) Dot plot of the expression of Piezo1 in 11 cell clusters in the callus on 14th day post-fracture. C, D) Representative μCT images and quantitative analysis of bone mass of femurs from 12-week male Piezo1WT and Piezo1Δ Lysm male mice (n = 6). E, F) Repre sentative μCT images of fractured femurs and quantitative analysis of callus from 12-week male Piezo1WT and Piezo1Δ Lysm male mice on 14th day post-surgery (n = 6). G, H) Representative images of HE and SO/FG staining in the fractured site from Piezo1WT and Piezo1Δ Lysm male mice. Scale bar: 500 μ m. I-L) Representative IF staining and quantitate analysis of RUNX2, OCN expression in the callus of Piezo1WT and Piezo1Δ Lysm male mice on 14th day post-fracture (n = 5). Scale bar: 100 μm. M, N) Representative IF staining and quantitate analysis of the type H vessels areas in the callus of Piezo1WT and Piezo1Δ Lysm male mice on 14th day post-fracture (n = 5). Scale bar: 100 μm. An individual sample was represented by a dot plot, and data are presented as means ± standard deviation (SD). *p < 0.05, **p < 0.01 and ***p < 0.001, ns: not significant difference. Statistical significance was determined by two-way ANOVA.

Article Snippet: The antibodies were as follows: RUNX2 (Abcam, ab192256; 1:1000), OCN (Takara, M188, 1:150), F4/80 (Invitrogen, 14–4801-82; 1:100), PIEZO1 (Proteintech, 15939–1-AP; 1:400), CD63 (Abcam, ab217345; 1:100), CD31 (R&D Systems, AF3628; 1:100) and Endomucin (Santa Cruz, sc-65495; 1:100), Ki67 (Abcam, ab15580; 1:1000), ADRB2 (Abcam, ab182136; 1:100).

Techniques: Expressing, Staining, Standard Deviation

Journal: Chemical Engineering Journal

Article Title: Extracellular vesicles from mechanical loading stimulated-macrophages favor fracture healing through targeting Adrb2 of osteoblasts

doi: 10.1016/j.cej.2024.159079

Figure Lengend Snippet: Fig. 4. PIEZO1 is essential for osteogenesis of ML-Mφ-sEVs. A) Representative image of the morphology of Mφ-sEVs and ML-Mφ-sEVs. Scale bar: 100 nm. B) Diameter of the distribution of Mφ-sEVs and ML-Mφ-sEVs. C) Flow cytometry analysis of the sEVs markers: CD9 and CD81. D) Representative ICC staining of the intake of PKH67-labeled sEVs by MC3T3-E1 cells. Scale bar: 50 μm. E) Representative ALP and Alizarin red staining of MC3T3-E1 cells treated with solvent, Mφ-sEVs, ML-Mφ-sEVs, or ML-MφPiezo1-KO -sEVs. Scale bar: 200 μm. F, G) Quantitative analysis of the ALP reactivity and mineralized area of MC3T3-E1 cells (n = 4). H) Quantitative analysis of mRNA expression of Runx2, Bglap, Alpl in MC3T3-E1 cells. The expression of Gapdh serves as the loading control (n = 3). I, J) Representative ICC staining images and quantitative analysis of RUNX2 and OCN expression in MC3T3-E1 cells (n = 4). Scale bar: 100 μm. K, L) Representative western blots images and quantitative analysis of RUNX2, OCN, and ALP expression in MC3T3-E1 cells. The expression of GAPDH serves as the loading control (n = 3). An individual sample was represented by a dot plot, and data are presented as means ± standard deviation (SD). *p < 0.05, **p < 0.01 and ***p < 0.001, ns: not significant difference. Statistical significance was determined by one-way ANOVA.

Article Snippet: The antibodies were as follows: RUNX2 (Abcam, ab192256; 1:1000), OCN (Takara, M188, 1:150), F4/80 (Invitrogen, 14–4801-82; 1:100), PIEZO1 (Proteintech, 15939–1-AP; 1:400), CD63 (Abcam, ab217345; 1:100), CD31 (R&D Systems, AF3628; 1:100) and Endomucin (Santa Cruz, sc-65495; 1:100), Ki67 (Abcam, ab15580; 1:1000), ADRB2 (Abcam, ab182136; 1:100).

Techniques: Flow Cytometry, Staining, Labeling, Solvent, Expressing, Control, Western Blot, Standard Deviation

Journal: Chemical Engineering Journal

Article Title: Extracellular vesicles from mechanical loading stimulated-macrophages favor fracture healing through targeting Adrb2 of osteoblasts

doi: 10.1016/j.cej.2024.159079

Figure Lengend Snippet: Fig. 5. ML-Mφ-sEVs promote fracture healing in vivo. A, B) Representative image of metabolic kinetics of Mφ-sEVs, ML-Mφ-sEVs, and ML-MφPiezo1-KO -sEVs in fracture sites after in situ injection on 7th day after fracture. C) Schematic graph of solvent, Mφ-sEVs, ML-Mφ-sEVs, and ML-MφPiezo1-KO -sEVs administration to the fracture site. D, E) Representative μCT images of fractured femurs and quantitative analysis of callus from 12-week male mice treated with solvent or sEVs (n = 5). F, G) Representative images of HE and SO/FG staining in the fractured site. Scale bar: 500 μ m. H-K) Representative images of IF staining and quantitate analysis of RUNX2 and OCN expression in the callus (n = 5). Scale bar: 100 μm. An individual sample was represented by a dot plot, and data are presented as means ± standard deviation (SD). *p < 0.05, **p < 0.01 and ***p < 0.001, ns: not significant difference. Statistical significance was determined by one-way ANOVA.

Article Snippet: The antibodies were as follows: RUNX2 (Abcam, ab192256; 1:1000), OCN (Takara, M188, 1:150), F4/80 (Invitrogen, 14–4801-82; 1:100), PIEZO1 (Proteintech, 15939–1-AP; 1:400), CD63 (Abcam, ab217345; 1:100), CD31 (R&D Systems, AF3628; 1:100) and Endomucin (Santa Cruz, sc-65495; 1:100), Ki67 (Abcam, ab15580; 1:1000), ADRB2 (Abcam, ab182136; 1:100).

Techniques: In Vivo, In Situ, Injection, Solvent, Staining, Expressing, Standard Deviation

Journal: Chemical Engineering Journal

Article Title: Extracellular vesicles from mechanical loading stimulated-macrophages favor fracture healing through targeting Adrb2 of osteoblasts

doi: 10.1016/j.cej.2024.159079

Figure Lengend Snippet: Fig. 7. Adrb2 of osteoblasts could be a target of bone repair. A) Schematic diagram of putative binding sites in the Adrb2-3′UTR and quantitative analysis of relative luciferase activity of recipient cells after mutating (MUT) the binding site of 3′-UTR-driven reporters (n = 4). B) Quantitative analysis of mRNA expression of Adrb2 in MC3T3-E1 cells transfected with mimic-NC, mimic-let-7 g-5p, inhibitor-NC, inhibitor-let-7 g-5p. The expression of Gapdh serves as the loading control (n = 4). C) Quantitative analysis of protein levels of ADRB2 in MC3T3-E1 cells transfected with mimic-NC, mimic-let-7 g-5p, inhibitor-NC, inhibitor-let-7 g-5p. The expression of GAPDH serves as the loading control (n = 4). D, E) Representative μCT images of fractured femurs and quantitative analysis of callus from 12-week male Adrb2WT and Adrb2Δ OCN male mice treated with NC– antagomir or let-7 g-5p antagomir on 14th day post-fracture (n = 6). F, G) Representative images of HE and SO/FG staining in the fractured site from 12-week Adrb2WT and Adrb2Δ OCN male mice. Scale bar: 500 μ m. H, I) Representative images of IHC staining and quantitate analysis of RUNX2 and OCN expression in the callus from 12-week male Adrb2WT and Adrb2Δ OCN male mice on 14th day post-fracture (n = 5). Scale bar: 50 μm. An individual sample was represented by a dot plot, and data are presented as means ± standard deviation (SD). *p < 0.05, **p < 0.01 and ***p < 0.001, ns: not significant. Statistical significance was determined by two-tailed Student’s t-test (A), one-way ANOVA (B and C), or two-way ANOVA (E to I).

Article Snippet: The antibodies were as follows: RUNX2 (Abcam, ab192256; 1:1000), OCN (Takara, M188, 1:150), F4/80 (Invitrogen, 14–4801-82; 1:100), PIEZO1 (Proteintech, 15939–1-AP; 1:400), CD63 (Abcam, ab217345; 1:100), CD31 (R&D Systems, AF3628; 1:100) and Endomucin (Santa Cruz, sc-65495; 1:100), Ki67 (Abcam, ab15580; 1:1000), ADRB2 (Abcam, ab182136; 1:100).

Techniques: Binding Assay, Luciferase, Activity Assay, Expressing, Transfection, Control, Staining, Immunohistochemistry, Standard Deviation, Two Tailed Test

Journal: Chemical Engineering Journal

Article Title: Extracellular vesicles from mechanical loading stimulated-macrophages favor fracture healing through targeting Adrb2 of osteoblasts

doi: 10.1016/j.cej.2024.159079

Figure Lengend Snippet: Fig. 9. An ultraviolet light-sensitive injectable hydrogel carrying engineered sEVs promoted fracture healing through targeting ADRB2 in osteoblasts. A) Schematic graph of the construction process of Gel + OBT-sEVlet-7g-5p and the application in vitro and vivo. B) Representative images of flow cytometry plots that demonstrate the delivering efficiency of osteoblast targeting peptide-modified sEVs to MC3T3-E1 cells. The OBT or control peptide was labeled with FITIC fluorophore. C) Representative images of ultraviolet curing effect of hydrogel and the administration to the fracture site. D) Representative images of SEM manifesting the internal ultrastructure of the gel and sEVs loading efficiency. Scale bar: 100 μ m; 500 nm. E) Quantitative analysis of the controlled release kinetics of OBT-sEVNC and OBT- sEVlet-7g-5p from hydrogel (n = 3). F) Representative μCT images of fractured femurs and quantitative analysis of callus from 12-week male mice treated with Gel, Gel + OBT, Gel + OBT-sEVNC or Gel + OBT-sEVlet-7g-5p (n = 5). G, H) Representative images of HE and SO/FG staining in the fractured site from 12-week male mice. Scale bar: 500 μ m. I, J) Representative IF staining of RUNX2, OCN expression in the callus of 12-week male mice on 14th day post-fracture (n = 5). Scale bar: 100 μm. K) Representative IF staining of ADRB2 expression in osteoblasts in the callus of 12-week male mice on 14th day post-fracture (n = 5). Scale bar: 100 μm. An individual sample was represented by a dot plot, and data are presented as means ± standard deviation (SD). *p < 0.05, **p < 0.01 and ***p < 0.001, ns: not significant difference. Statistical significance was determined by one-way ANOVA.

Article Snippet: The antibodies were as follows: RUNX2 (Abcam, ab192256; 1:1000), OCN (Takara, M188, 1:150), F4/80 (Invitrogen, 14–4801-82; 1:100), PIEZO1 (Proteintech, 15939–1-AP; 1:400), CD63 (Abcam, ab217345; 1:100), CD31 (R&D Systems, AF3628; 1:100) and Endomucin (Santa Cruz, sc-65495; 1:100), Ki67 (Abcam, ab15580; 1:1000), ADRB2 (Abcam, ab182136; 1:100).

Techniques: In Vitro, Flow Cytometry, Modification, Control, Labeling, Staining, Expressing, Standard Deviation