Journal: Frontiers in Physiology

Article Title: Sex Differences in Metabolic and Behavioral Responses to Exercise but Not Exogenous Osteocalcin Treatment in Mice Fed a High Fat Diet

doi: 10.3389/fphys.2022.831056

Figure Lengend Snippet: Glucose tolerance tests. At 12 weeks of age after 8 weeks of control diet (Control) or high fat diet (HFD); (A) male, (B) female time-courses, (C) area under the curve. Mice at 20 weeks of age following 8 weeks of treatments (D–F) , sedentary mice on control diet with daily saline injection (Control), sedentary mice on HFD (HFD Sed Sal), exercised mice on HFD (HFD Ex Sal) or sedentary mice on HFD treated with osteocalcin (HFD Sed Ocn). (D) Male mice, (E) female time-courses, and (F) area under the curve. Error bars represent standard deviation from the mean; 8-week GTT male groups n = 6, female groups n = 5; end of treatment GTT Male groups n = 5, female groups n = 7. *Indicates significant differences after Bonferroni correction, unadjusted p values are shown above bars.

Article Snippet: Serum mouse osteocalcin concentrations were determined using ELISAs specific to mouse carboxylated osteocalcin (Gla-OC MK127, Takara Bio United States, Mountain View, CA, United States) and uncarboxylated osteocalcin (Glu-OC MK129, Takara Bio).

Techniques: Injection, Standard Deviation

Journal: Frontiers in Physiology

Article Title: Sex Differences in Metabolic and Behavioral Responses to Exercise but Not Exogenous Osteocalcin Treatment in Mice Fed a High Fat Diet

doi: 10.3389/fphys.2022.831056

Figure Lengend Snippet: Metabolic measures after 8 weeks of treatments.

Article Snippet: Serum mouse osteocalcin concentrations were determined using ELISAs specific to mouse carboxylated osteocalcin (Gla-OC MK127, Takara Bio United States, Mountain View, CA, United States) and uncarboxylated osteocalcin (Glu-OC MK129, Takara Bio).

Techniques:

Journal: Frontiers in Physiology

Article Title: Sex Differences in Metabolic and Behavioral Responses to Exercise but Not Exogenous Osteocalcin Treatment in Mice Fed a High Fat Diet

doi: 10.3389/fphys.2022.831056

Figure Lengend Snippet: Progressive problem-solving in puzzle box. Latencies for males and females of each treatment group during the puzzle box test (A–D) squares represent data from males, triangles represent data from females. (A) Controls; (B) High fat diet, sedentary with daily saline injections (HFD Sed Sal); (C) High fat diet, daily exercise and saline injections (HFD Ex Sal); (D) High fat diet sedentary with daily osteocalcin injections (HFD Sed Ocn); (E) Problem-solving scores, Z-normalized to the male control group. Data point and bars represent mean ± standard deviation; male control n = 10, HFD groups n = 9; female control n = 10, HFD n = 12. Statistically significant differences after Bonferroni correction indicated by * with unadjusted p values shown above bars.

Article Snippet: Serum mouse osteocalcin concentrations were determined using ELISAs specific to mouse carboxylated osteocalcin (Gla-OC MK127, Takara Bio United States, Mountain View, CA, United States) and uncarboxylated osteocalcin (Glu-OC MK129, Takara Bio).

Techniques: Standard Deviation

Journal: Stem cell research & therapy

Article Title: Methylsulfonylmethane enhances MSC chondrogenic commitment and promotes pre-osteoblasts formation.

doi: 10.1186/s13287-021-02396-5

Figure Lengend Snippet: Fig. 4 Effects of MSM in the osteogenic maturation. RUNX2 gene expression was reduced while the expression of SPARC and SPP1 (A), as well the number of osteocalcin positive cells (B) increased in MSM-treated cells after 14 days of osteogenic differentiation. After 21 days of differentiation calcium deposition, evaluated by alizarin red staining (ARS), increased in cells treated with MSM (C). *p<0.05; **p<0.01

Article Snippet: Primary antibodies for RUNX2 and osteocalcin (sc74495, Santa Cruz, Dallas, Texas, USA) were diluted according to the manufacturer’s instruction in antibody dilution buffer and incubated overnight at 4°C.

Techniques: Gene Expression, Expressing, Staining

Journal: PLoS Genetics

Article Title: WNT7B Promotes Bone Formation in part through mTORC1

doi: 10.1371/journal.pgen.1004145

Figure Lengend Snippet: (A, B) H&E staining of longitudinal tibia sections from two-month-old control (A) or ColI-Wnt7b littermates (B). 1°, 2°: primary and secondary ossification center. Shown to the right are higher magnification images of secondary ossification center (A1, B1), growth plate (A2, B2), primary spongiosa (A3, B3), and marrow region (A4, B4). Scale bar: 0.5 mm in panels A, B; 0.1 mm in panels A1–A4, B1–B4. (C) Serum osteocalcin levels of control (C) and ColI-Wnt7b littermates (7b) at one and two months of age. (D) Number of osteoblasts normalized to trabecular bone perimeter on longitudinal tibia sections. (E–F) Representative images of calcein double labeling in the humerus of two-month-old control (E) and ColI-Wnt7b (F) littermates. (G) Dynamic histomorphometry parameters from secondary ossification center of the humerus. MAR: mineral apposite rate; MS/BS: mineralizing surface over bone surface; BFR/BS: bone formation rate. (H) Serum CTX-I levels. (I) Osteoclast parameters from histomorphometry. #Oc/mm: osteoclast number normalized to trabecular bone perimeter, µm/Oc (average osteoclast surface), Oc S/BS (osteoclast surface normalized to bone surface). All bar graphs show mean ± STDEV, *: P<0.05, n = 3.

Article Snippet: Serum osteocalcin levels were determined with the Mouse Osteocalcin EIA Kit (Biomedical Technologies, Stoughton, MA).

Techniques: Staining, Control, Labeling

Journal: Biomedicines

Article Title: Osteocalcin Alleviates Lipopolysaccharide-Induced Acute Inflammation via Activation of GPR37 in Macrophages

doi: 10.3390/biomedicines10051006

Figure Lengend Snippet: OCN attenuates LPS-caused detrimental effects in WT and OCN −/− mice. ( A ) Experimental design for the LPS-induced acute inflammation. The OCN −/− and WT mice were injected intraperitoneally with PBS or OCN (500 ng) followed by LPS (10 mg/kg) treatment. Mice survival rate and serum inflammatory were analyzed in each time point, accordingly. ( B ) Survival curves of OCN −/− and WT mice treated with PBS or OCN in response to LPS stimulation. Sample sizes are presented in brackets ( n = 15). * p < 0.05, as compared to PBS. ( C , D ) ELISA analysis of serum levels of IL-6 ( C ) and TNFα ( D ) in OCN −/− and WT mice with PBS or OCN pretreatment after 6h of LPS administration. Sample sizes are indicated as dots in each column. Data are presented as mean ± SD. * p < 0.05 and ** p < 0.01, as compared to PBS.

Article Snippet: Mouse osteocalcin (OCN) peptides were synthesized in the Beijing Genomics Institute (BGI, Beijing, China).

Techniques: Injection, Enzyme-linked Immunosorbent Assay

Journal: Biomedicines

Article Title: Osteocalcin Alleviates Lipopolysaccharide-Induced Acute Inflammation via Activation of GPR37 in Macrophages

doi: 10.3390/biomedicines10051006

Figure Lengend Snippet: OCN inhibits the production of pro-inflammatory factor in macrophage. ( A , B ) Effects of OCN treatment on the mRNA expression of pro-inflammatory genes, i.e., IL-6 ( A ) and TNFα ( B ), in LPS (500 ng/mL) treated peritoneal macrophages from WT mice. Data are presented as means ± SD ( n = 3 biological replicates). * p < 0.05 and ** p < 0.01, as compared to PBS. ( C , D ) Cytokine levels in a culture medium of peritoneal macrophage from WT and OCN −/− mice. Peritoneal macrophages were treated with LPS (500 ng/mL), together PBS or OCN (10 nM) for 12 h, and the levels of IL-6 ( C ) and TNFα ( D ) in culture medium were measured by ELISA analysis. Data are presented as means ± SD. ( n = 3 biological replicates) * p < 0.05, as compared to PBS. ( E ) The mRNA expression analysis of genes associated with anti-inflammatory in peritoneal macrophages isolated from WT mice. Data are presented as means ± SD ( n = 3 biological replicates). ** p < 0.01 and *** p < 0.001, as compared to PBS.

Article Snippet: Mouse osteocalcin (OCN) peptides were synthesized in the Beijing Genomics Institute (BGI, Beijing, China).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Isolation

Journal: Biomedicines

Article Title: Osteocalcin Alleviates Lipopolysaccharide-Induced Acute Inflammation via Activation of GPR37 in Macrophages

doi: 10.3390/biomedicines10051006

Figure Lengend Snippet: GPR37 mediates OCN-induced intracellular responses in macrophages. ( A ) Overview of the experimental design testing intracellular signals in macrophages. Peritoneal macrophages were isolated from WT and GPR37 −/− mice to study the changes of intracellular calcium level (iCa 2+ ), cAMP production, and pERK level in response to OCN treatment. ( B ) Representative response curves of OCN-triggered iCa 2+ changes in macrophages from WT and GPR37 −/− mice. ( C ) OCN-triggered inhibition of cAMP production in macrophages from WT and GPR37 −/− mice. Data are presented as mean ± SD ( n = 3 biological replicates). * p < 0.05, as compared to PBS, and n.s. indicates not significant. ( D , E ) Representative immunoblot images ( D ) and relative quantification ( E ) showing that OCN-triggered an increase in the pERK level in macrophages from WT but not in that from GPR37 −/− mice. Data are presented as mean ± SD ( n = 3 biological replicates). * p < 0.05, as compared to PBS. ( F ) Representative response curves of OCN-triggered iCa 2+ changes in PTX pretreated macrophage from WT mice. ( G ) OCN-triggered inhibition of cAMP production in PTX pretreated macrophage from WT mice. Data are presented as mean ± SD ( n = 3 biological replicates). * p < 0.05, as compared to PBS, and n.s. indicates not significant. ( H , I ) Representative immunoblot images ( H ) and relative quantification ( I ) showing that PTX pretreatment blocked the OCN-triggered increase in the pERK level in macrophages from WT mice. Data are presented as mean ± SD ( n = 3 biological replicates). * p < 0.05, as compared to PBS.

Article Snippet: Mouse osteocalcin (OCN) peptides were synthesized in the Beijing Genomics Institute (BGI, Beijing, China).

Techniques: Isolation, Inhibition, Western Blot, Quantitative Proteomics

Journal: Biomedicines

Article Title: Osteocalcin Alleviates Lipopolysaccharide-Induced Acute Inflammation via Activation of GPR37 in Macrophages

doi: 10.3390/biomedicines10051006

Figure Lengend Snippet: GPR37 mediates the inhibitory effects of OCN on pro-inflammatory factors in macrophage. ( A , B ) Cytokine levels in the culture medium of peritoneal macrophages from WT and GPR37 −/− mice. Cells were treated with LPS (500 ng/mL, 37 °C, 24 h) together with PBS or OCN (10 nM). ELISA analysis was performed to measure the level of IL-6 ( A ) and TNF-α ( B ). Data are presented as mean ± SD ( n = 3 biological replicates), * p < 0.05, as compared to PBS, and n.s. indicates not significant. ( C , D ) Representative immunoblot images ( C ) and quantitative analysis ( D ) of NFκB p65 level in macrophages treated with LPS in the presence of OCN. Data are presented as mean ± SD ( n = 3 biological replicates), * p < 0.05 as compared to PBS, and n.s. indicates not significant.

Article Snippet: Mouse osteocalcin (OCN) peptides were synthesized in the Beijing Genomics Institute (BGI, Beijing, China).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Biomedicines

Article Title: Osteocalcin Alleviates Lipopolysaccharide-Induced Acute Inflammation via Activation of GPR37 in Macrophages

doi: 10.3390/biomedicines10051006

Figure Lengend Snippet: OCN promotes macrophage phagocytosis via GPR37. ( A ) Representative images of in vitro phagocytosis assay, in which pHrodo zymosan particles were incubated with peritoneal macrophages from WT or OCN −/− mice, treated with PBS or OCN (10 nM, 30 min, 37 °C). Red fluorescence suggests an intracellular update indicating phagocytosis. Scale bars: 20 μm. ( B ) Quantification of macrophage phagocytic activity of zymosan positives cells. Data are presented as mean ± SD ( n = 3 biological replicates), ** p < 0.01, as compared to PBS. ( C ) Representative images of an in vitro phagocytosis assay, in which pHrodo zymosan particles were incubated with peritoneal macrophages from WT or GPR37 −/− mice, treated with PBS or OCN (10 nM, 30 min, 37 °C). Scale bars: 20 μm. ( D ) Quantification of macrophage phagocytic activity of zymosan positives cells. Data are presented as mean ± SD ( n = 3 biological replicates), ** p < 0.01, as compared to PBS, and n.s. indicates not significant.

Article Snippet: Mouse osteocalcin (OCN) peptides were synthesized in the Beijing Genomics Institute (BGI, Beijing, China).

Techniques: In Vitro, Phagocytosis Assay, Incubation, Fluorescence, Activity Assay

Journal: Biomedicines

Article Title: Osteocalcin Alleviates Lipopolysaccharide-Induced Acute Inflammation via Activation of GPR37 in Macrophages

doi: 10.3390/biomedicines10051006

Figure Lengend Snippet: The protective function of OCN against LPS is absent in GPR37 −/− mice. ( A ) Experimental design for LPS-induced acute inflammation in GPR37 −/− and WT mice. After intraperitoneal injection of PBS or OCN (500 ng), followed by LPS (10 mg/kg) treatment, mice survival rate and serum inflammatory cytokine level were analyzed accordingly. ( B ) Survival curves of GPR37 −/− and WT mice treated with PBS or OCN in response to an LPS challenge. Sample sizes are presented in brackets ( n = 15). * p < 0.05, as compared to PBS. ( C , D ) After 6h of LPS injection, with or without OCN pretreatment, serum IL-6 ( C ) and TNFα ( D ) levels were analyzed in GPR37 −/− and WT mice. Sample sizes are indicated as dots in columns. * p < 0.05 and ** p < 0.01, as compared to PBS; n.s. indicates not significant.

Article Snippet: Mouse osteocalcin (OCN) peptides were synthesized in the Beijing Genomics Institute (BGI, Beijing, China).

Techniques: Injection

Journal: Biomedicines

Article Title: Osteocalcin Alleviates Lipopolysaccharide-Induced Acute Inflammation via Activation of GPR37 in Macrophages

doi: 10.3390/biomedicines10051006

Figure Lengend Snippet: Adoptive transfer of OCN-treated macrophages confers protection against LPS stimulation. ( A ) Experimental design, to test whether adoptive transfer of macrophages pretreated with OCN, could attenuate LPS-induced acute inflammation. Peritoneal macrophages were isolated from WT or GPR37 −/− mice and then treated with PBS or OCN (10 nM) for 24 h, followed by a washout of OCN and an adoptive transfer of macrophages (I.P.) into GPR37 −/− mice challenged with LPS for 1 h. ( B , C ) ELISA analysis of serum IL-6 ( B ) and TNFα ( C ) levels, in GPR37 −/− mice with adoptive transfer, of macrophages derived from WT or GPR37 −/− mice. Sample sizes are indicated as dots in each column. Data are presented as mean ± SD. * p < 0.05 and ** p < 0.01, as compared to PBS; n.s. indicates not significant.

Article Snippet: Mouse osteocalcin (OCN) peptides were synthesized in the Beijing Genomics Institute (BGI, Beijing, China).

Techniques: Adoptive Transfer Assay, Isolation, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Biomedicines

Article Title: Osteocalcin Alleviates Lipopolysaccharide-Induced Acute Inflammation via Activation of GPR37 in Macrophages

doi: 10.3390/biomedicines10051006

Figure Lengend Snippet: A schematic diagram illustrating that the bone-derived hormone OCN, via the activation of GPR37 in macrophages, plays a protective function against LPS challenge through the regulation of macrophage inflammatory reactions and phagocytic function.

Article Snippet: Mouse osteocalcin (OCN) peptides were synthesized in the Beijing Genomics Institute (BGI, Beijing, China).

Techniques: Derivative Assay, Activation Assay

Journal: Development (Cambridge, England)

Article Title: Osteogenic capillaries orchestrate growth plate-independent ossification of the malleus

doi: 10.1242/dev.123885

Figure Lengend Snippet: Histological analysis of the mPb. (A) Hematoxylin and eosin (H&E) staining of the mPb. Low and high magnifications are shown. Scale bars: 100 µm in upper panels, 20 µm in lower panels. (B) Immunofluorescence staining of the mPb for osteocalcin (Ocn, green). Sections were counterstained with DAPI (blue). Scale bars: 50 µm. (C) Horizontal section showing H&E staining of the mPb from a 12-week-old female mouse. Scale bar: 100 µm. (D) Phase contrast image corresponding to boxed area in C, with green endothelial cells (arrows) and orange osteocytes (arrowheads).

Article Snippet: Anti-mouse CD31 (PECAM-1) hamster monoclonal antibody (1:500, 2H8, Millipore), anti-mouse Fosl1 rabbit polyclonal antibody (1:50, N-17, Santa Cruz), anti-mouse osteocalcin polyclonal antibody (1:1000, ALX-210-333, Enzo), anti-mouse osteocalcin rat monoclonal antibody (1:200, R21C-01A, Takara), and anti-mouse endomucin rat monoclonal antibody (1:50, V.7C7, Santa Cruz) were used.

Techniques: Staining, Immunofluorescence

Journal: Development (Cambridge, England)

Article Title: Osteogenic capillaries orchestrate growth plate-independent ossification of the malleus

doi: 10.1242/dev.123885

Figure Lengend Snippet: Bone formation around capillaries. (A) Immunofluorescence indicating capillary endothelial cells (CD31-positive, green) and osteoblasts [osteocalcin (Ocn)-positive, red] in the mPb at P21. Scale bar: 50 µm. Right panel: higher magnification of boxed area in left panel. White dots outline malleus. Scale bar: 20 µm. (B) Alizarin Red/calcein double labeling of the mPb. Alizarin Red and calcein were injected at P25 and P28, respectively. Mice were sacrificed at P29. Scale bar: 50 µm. Right panel: higher magnification of boxed area in left panel. Scale bar: 20 µm. (C,D) Calcein was injected intraperitoneally at P21 into female C57BL/6 mice, and animals were analyzed 24 h later. Calcein (green) and endomucin (Emcn) (C, red) or osteocalcin (D, red) labeling in the mPb. White dotted lines in D encircle nuclei of endothelial cells. Scale bars: 20 µm.

Article Snippet: Anti-mouse CD31 (PECAM-1) hamster monoclonal antibody (1:500, 2H8, Millipore), anti-mouse Fosl1 rabbit polyclonal antibody (1:50, N-17, Santa Cruz), anti-mouse osteocalcin polyclonal antibody (1:1000, ALX-210-333, Enzo), anti-mouse osteocalcin rat monoclonal antibody (1:200, R21C-01A, Takara), and anti-mouse endomucin rat monoclonal antibody (1:50, V.7C7, Santa Cruz) were used.

Techniques: Immunofluorescence, Labeling, Injection

Journal: Development (Cambridge, England)

Article Title: Osteogenic capillaries orchestrate growth plate-independent ossification of the malleus

doi: 10.1242/dev.123885

Figure Lengend Snippet: Conditional Fosl1 overexpression antagonizes normal decreases in capillary volume in the mPb. Expression of Fosl1 (red) plus endomucin (Emcn) (A, green) or osteocalcin (Ocn) (B, green) in mPb isolated from 4-week-old male Fosl1 tetON ( Rosa26 -rtTA; TetOP -Fosl1) mice fed food containing dox from P28 to P31. Scale bars: 50 µm in left panel; 20 µm in right panel. White dots outline malleus. (C) Representative μCT images (voxel size, 1.4 µm) of capillaries in the mPb isolated from non-inducible control ( TetOP -Fosl1, top) and Fosl1-inducible Fosl1 tetON ( Rosa26 -rtTA; TetOP -Fosl1, bottom) mice. Mice of both genotypes were fed a dox-containing diet starting from P21 and sacrificed at P56. Scale bars: 100 µm. (D) H&E staining of the mPb in non-inducible control and Fosl1 tetON mice at P56. Capillary area is greater in Fosl1 tetON mice. Arrowheads indicate osteoblasts. Scale bars: 100 µm in left panels; 20 µm in right panels. (E-G) Quantification of capillary volume (E), number (F) and cross-sectional area of capillaries calculated in a plane 150 µm from the distal end (G) of the mPb. Control, n =5; Fosl1 tetON , n =4. ** P <0.01. Data are represented as means±s.e.m.

Article Snippet: Anti-mouse CD31 (PECAM-1) hamster monoclonal antibody (1:500, 2H8, Millipore), anti-mouse Fosl1 rabbit polyclonal antibody (1:50, N-17, Santa Cruz), anti-mouse osteocalcin polyclonal antibody (1:1000, ALX-210-333, Enzo), anti-mouse osteocalcin rat monoclonal antibody (1:200, R21C-01A, Takara), and anti-mouse endomucin rat monoclonal antibody (1:50, V.7C7, Santa Cruz) were used.

Techniques: Over Expression, Expressing, Isolation, Staining

Journal: The Journal of Cell Biology

Article Title: Clonal Isolation of Muscle-Derived Cells Capable of Enhancing Muscle Regeneration and Bone Healing

doi:

Figure Lengend Snippet: Morphologic change and expression of osteocalcin by mc13 cells with exposure to rhBMP-2. Mc13 cells were incubated in growth media without rhBMP-2 for 6 d. When cells became >50% confluent, they began to fuse and form multinucleated myotubes (A). When mc13 cells were incubated in growth media containing 200 ng/ml rhBMP-2, cells remained mononucleated and did not fuse (B). When cells reached >90% confluency without rhBMP-2, almost all the cells fused to form myotubes (C). With rhBMP-2, when cells reached >90% confluency, round, hypertrophic cells began to appear in the culture (D, arrows). These round, hypertrophic cells were highly positive for osteocalcin expression (E, arrows). Bar, 50 μm.

Article Snippet: The muscle sections were incubated with DAPI at a dilution of 1/100 in PBS to stain the nuclei; a biotinylated anti–β-galactosidase antibody (1/100 in PBS; Sigma-Aldrich), followed by streptavidin conjugated to fluorescein (Gal-13, 1/300 in PBS; Sigma-Aldrich) to stain the β-galactosidase expressing nuclei; and a goat anti–mouse osteocalcin (1:100 in PBS; Chemicon Co), followed by an incubation with a Cy3-conjugated anti-goat antibody (1/100 in PBS; Sigma-Aldrich) to label the osteocalcin expressing cells.

Techniques: Expressing, Incubation

Journal: The Journal of Cell Biology

Article Title: Clonal Isolation of Muscle-Derived Cells Capable of Enhancing Muscle Regeneration and Bone Healing

doi:

Figure Lengend Snippet: In vivo differentiation of mc13 cells into osteogenic lineage after genetic engineering to express BMP-2. The amount of BMP-2 secreted by the mc13 cells that were transduced with adBMP-2 was found significantly higher (* P < 0.05) than the nontransduced mc13 cells (A). 0.5–1.0 × 10 6 cells genetically engineered to express BMP-2 were injected into hind limbs of SCID mice. After 14 d, mice were killed, and the hind limb muscle tissues were analyzed radiographically for evidence of bone formation. There was a robust ectopic bone formation (seen radiographically) within skeletal muscle in all mice injected with mc13 cells transduced with adBMP-2 (B, arrow). The injected muscle containing the ectopic bone was then harvested and stained for β-galactosidase activity to locate injected cells. The LacZ positive cells were uniformly found within the lacunae, a location where osteoblasts and osteocytes are normally found (C). The ectopic bone was also stained for presence of dystrophin. As indicated by green fluorescence, the ectopic bone contained abundant cells expressing dystrophin, confirming that mc13 cells were active participants in formation of bone (D). To determine whether the genetically engineered mc13 expressing BMP-2 can express bone protein, we colocalized β-galactosidase expressing nuclei, osteocalcin expression, and nuclei staining (DAPI) by immunohistochemistry (E–H). We identified nuclei expressing β-galactosidase (see F, arrow, FITC/green) that expressed osteocalcin (see G, arrow, cy3/red) and colocalized with nuclei staining (see E, arrow, DAPI/blue). The triple colocalization of DAPI/osteocalcin and β-galactosidase ( H, arrows) suggests that the genetically engineered mc13 can express bone protein (osteocalcin). We have also observed β-galactosidase expressing nuclei ( F, arrowhead) that were not colocalized with osteocalcin expressing cells ( G, arrowhead), suggesting that some of the engineered mc13 were not expressing osteocalcin ( , E–H, arrowheads). Bar: (C and D) 50 μm; (E–H) 25 μm.

Article Snippet: The muscle sections were incubated with DAPI at a dilution of 1/100 in PBS to stain the nuclei; a biotinylated anti–β-galactosidase antibody (1/100 in PBS; Sigma-Aldrich), followed by streptavidin conjugated to fluorescein (Gal-13, 1/300 in PBS; Sigma-Aldrich) to stain the β-galactosidase expressing nuclei; and a goat anti–mouse osteocalcin (1:100 in PBS; Chemicon Co), followed by an incubation with a Cy3-conjugated anti-goat antibody (1/100 in PBS; Sigma-Aldrich) to label the osteocalcin expressing cells.

Techniques: In Vivo, Transduction, Injection, Staining, Activity Assay, Fluorescence, Expressing, Immunohistochemistry

Journal:

Article Title: AN ELISA-BASED METHOD TO QUANTIFY OSTEOCALCIN CARBOXYLATION IN MICE

doi: 10.1016/j.bbrc.2010.06.008

Figure Lengend Snippet: (A) The specificity of the different goat antibodies were tested by western blotting on dot blot membranes. GLA-OCN: fully carboxylated osteocalcin. GLU-OCN: uncarboxylated osteocalcin.

Article Snippet: GLU, GLA13 and total mouse osteocalcin ELISA Antibody Coating Buffer (CB1), ELISA Wash Buffer (WB1), General Blocker Buffer (BB1), General Assay Diluent (AD1) and Stop Solution for TMB (STOP1) were all obtained from ImmunoChemistry Technologies.

Techniques: Western Blot, Dot Blot

Journal:

Article Title: AN ELISA-BASED METHOD TO QUANTIFY OSTEOCALCIN CARBOXYLATION IN MICE

doi: 10.1016/j.bbrc.2010.06.008

Figure Lengend Snippet: (A) Quantitative measurement of GLU-OCN, GLA13-OCN and total osteocalcin in the supernatant of primary osteoblasts cultures (pOB) treated with a vehicle or with warfarin. *** p < 0.001 compared to vehicle treated osteoblasts.

Article Snippet: GLU, GLA13 and total mouse osteocalcin ELISA Antibody Coating Buffer (CB1), ELISA Wash Buffer (WB1), General Blocker Buffer (BB1), General Assay Diluent (AD1) and Stop Solution for TMB (STOP1) were all obtained from ImmunoChemistry Technologies.

Techniques:

Journal:

Article Title: AN ELISA-BASED METHOD TO QUANTIFY OSTEOCALCIN CARBOXYLATION IN MICE

doi: 10.1016/j.bbrc.2010.06.008

Figure Lengend Snippet: (A) Quantitative measurement of GLU-OCN, GLA13-OCN and total osteocalcin in serums from 2 months old wild type (WT) and Ocn −/− mice. All three forms were undetectable (UN) in Ocn −/− serums.

Article Snippet: GLU, GLA13 and total mouse osteocalcin ELISA Antibody Coating Buffer (CB1), ELISA Wash Buffer (WB1), General Blocker Buffer (BB1), General Assay Diluent (AD1) and Stop Solution for TMB (STOP1) were all obtained from ImmunoChemistry Technologies.

Techniques: