Journal: Frontiers in Molecular Neuroscience

Article Title: Osteocalcin Ameliorates Motor Dysfunction in a 6-Hydroxydopamine-Induced Parkinson’s Disease Rat Model Through AKT/GSK3β Signaling

doi: 10.3389/fnmol.2018.00343

Figure Lengend Snippet: The levels of osteocalcin (OCN) in the serum or cerebrospinal fluid (CSF) of control and 6-hydroxydopamine (6-OHDA)-induced Parkinson’s disease (PD) rat models. The CSF OCN levels in PD rats dramatically decreased compared to control rats ( A , n = 17). No difference in the serum OCN levels was found between control and PD rats ( B , n = 19). Expression of GPR158 and GPRC6A in various brain-derived tissues of 8-week-old rats ( C , n = 3). Coimmunoprecipitation of 8-week-old mice striata and proteins were then subjected to western blot analysis using anti-Gpr158 and anti-GPRC6a antibodies ( D , n = 3). Values were obtained from three independent experiments and results are given as the mean ± SEM. * P < 0.05.

Article Snippet: A rat OCN ELISA kit was purchased from MyBioSource (San Diego, CA, USA, Catalog: MBS2022619).

Techniques: Control, Expressing, Derivative Assay, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: Osteocalcin Ameliorates Motor Dysfunction in a 6-Hydroxydopamine-Induced Parkinson’s Disease Rat Model Through AKT/GSK3β Signaling

doi: 10.3389/fnmol.2018.00343

Figure Lengend Snippet: Effect of intraperitoneal (i.p.) injection of OCN on distance traveled (A) and rearing responses (B) in the open-field test (OFT). The cylinder test (C) and elevated body swing test (EBST; D ) were used to evaluate locomotor asymmetry. Compared with the sham groups, 6-OHDA injection decreased the distance traveled by rats in the OFT, and reduced the use of contralateral (left) forelimb in the cylinder test. The injection of 4 μg/kg OCN significantly improved the movement distance of 6-OHDA-lesioned rats in the 4th week ( P < 0.001), while 40 μg/kg OCN increased the number of rearing responses in the 3rd week ( P < 0.05). The representative traces of the OFT are shown in the lower panel of (A,B) . In the cylinder test, 4 μg/kg OCN improved the use of the contralateral forelimb ( P < 0.05). No obvious differences were observed in the EBST. n = 8–10, * P < 0.05 (compared with the 6-OHDA group).

Article Snippet: A rat OCN ELISA kit was purchased from MyBioSource (San Diego, CA, USA, Catalog: MBS2022619).

Techniques: Injection

Journal: Frontiers in Molecular Neuroscience

Article Title: Osteocalcin Ameliorates Motor Dysfunction in a 6-Hydroxydopamine-Induced Parkinson’s Disease Rat Model Through AKT/GSK3β Signaling

doi: 10.3389/fnmol.2018.00343

Figure Lengend Snippet: Effect of intra-striatum injection of OCN on distance traveled (A) , rearing responses (B) in OFT. The cylinder test (C) and EBST (D) were used to evaluate locomotor asymmetry. Compared with the sham groups, 6-OHDA injection decreased the distance traveled by rats, while the injection of OCN (4 μg) or nerve growth factor (NGF) significantly improved movement distance (A) , but not rearing impairment (B) of 6-OHDA-lesioned rats in OFT. The representative traces of the OFT are shown in the lower panel of (B) . No obvious differences were observed in the cylinder test (C) or the EBST (D) . n = 9–11, * P < 0.05 (compared with the 6-OHDA group).

Article Snippet: A rat OCN ELISA kit was purchased from MyBioSource (San Diego, CA, USA, Catalog: MBS2022619).

Techniques: Injection

Journal: Frontiers in Molecular Neuroscience

Article Title: Osteocalcin Ameliorates Motor Dysfunction in a 6-Hydroxydopamine-Induced Parkinson’s Disease Rat Model Through AKT/GSK3β Signaling

doi: 10.3389/fnmol.2018.00343

Figure Lengend Snippet: The protective effect of OCN on tyrosine hydroxylase (TH + ) neurons and fibers in the nigrostriatal system in the unilateral 6-OHDA-lesioned PD rat model. 6-OHDA induced ~90% dopaminergic neuronal loss on the lesioned side compared to the intact side of the substantia nigra (SN), which was improved by i.p. OCN injection (A) . Similarly, the decrease in TH-positive cell number in the SN of the lesioned side was also significantly alleviated by OCN injection (B) . The results from western blots measuring TH were consistent with immunohistochemistry in the striatum (C) . In the intra-striatum injection OCN group, 6-OHDA induced ~50% dopaminergic neuronal loss in the lesioned side compared to the intact side of the SN, which was improved by OCN injection (D) . Similarly, the decrease in TH-positive cell number in the SN of lesioned side was also significantly alleviated by intra-striatum OCN injection (E) . The results from western blots measuring TH were consistent with immunohistochemistry in the striatum (F) . n = 16, * P < 0.05 (compared with the 6-OHDA group).

Article Snippet: A rat OCN ELISA kit was purchased from MyBioSource (San Diego, CA, USA, Catalog: MBS2022619).

Techniques: Injection, Western Blot, Immunohistochemistry

Journal: Frontiers in Molecular Neuroscience

Article Title: Osteocalcin Ameliorates Motor Dysfunction in a 6-Hydroxydopamine-Induced Parkinson’s Disease Rat Model Through AKT/GSK3β Signaling

doi: 10.3389/fnmol.2018.00343

Figure Lengend Snippet: The effect of OCN on the proliferation of astrocytes and microglia. 6-OHDA significantly increased the number of GFAP-positive astrocytes in the lesioned side compared to the intact side both in the SN (A) and striatum (B) , which was inhibited by OCN injection. The same function of OCN on microglial proliferation was also observed, which was stained with Iba1 (C,D) . n = 16. Green = TH, Red = GFAP or Iba1, Blue = Nucleus.

Article Snippet: A rat OCN ELISA kit was purchased from MyBioSource (San Diego, CA, USA, Catalog: MBS2022619).

Techniques: Injection, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Osteocalcin Ameliorates Motor Dysfunction in a 6-Hydroxydopamine-Induced Parkinson’s Disease Rat Model Through AKT/GSK3β Signaling

doi: 10.3389/fnmol.2018.00343

Figure Lengend Snippet: Neuroprotective effects of OCN on 6-OHDA-induced neurotoxicity. PC12 cells were incubated with different concentrations of 6-OHDA for 24 h, and cell viability was measured by cell counting kit-8 (CCK-8; A ). Pretreatment of PC12 cells with various concentrations of OCN (1–100 ng/ml) was performed 2 h before exposure to 100 μM 6-OHDA, and the cell viability was measured at 24 h (B) . The effects of OCN on the auto-oxidation of 6-OHDA were measured spectrophotometrically at 490 nm using a microplate reader (C) . The results from western blots showed that treatment with 100 μM 6-OHDA increased the expression of caspase-3 and decreased the expression of bcl-2, and both changes were reversed by pre-incubation with OCN (D) . 6-OHDA decreased the expression of phosphorylated AKT (Ser473) and phosphorylated GSK3β (Ser9) at 24 h (E) , and both reductions were reversed by pretreatment with OCN (F) . Similarly, 6-OHDA injection into the right striatum also resulted in decreased expression of p-AKT and p-GSK3β, and both reductions were inhibited by direct injection of OCN into the striatum (G) . Values were obtained from three independent experiments. * P < 0.05 (compared with the control group), # P < 0.05 (compared with the 6-OHDA group).

Article Snippet: A rat OCN ELISA kit was purchased from MyBioSource (San Diego, CA, USA, Catalog: MBS2022619).

Techniques: Incubation, Cell Counting, CCK-8 Assay, Western Blot, Expressing, Injection, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: Osteocalcin Ameliorates Motor Dysfunction in a 6-Hydroxydopamine-Induced Parkinson’s Disease Rat Model Through AKT/GSK3β Signaling

doi: 10.3389/fnmol.2018.00343

Figure Lengend Snippet: OCN protects PC12 cells from 6-OHDA-induced neurotoxicity. Apoptotic cells were measured by flow cytometry (A–E) . The number of apoptotic cells was counted in the R2 (AV + /7-amino-actinomycin, 7-AAD + , the late phase apoptotic cells) and R4 (AV + /7-AAD − , the early phase apoptotic cells) regions, and the apoptotic rate was calculated (F) . The CCK-8 test showed that OCN could attenuate the neuronal toxicity induced by 6-OHDA through activating AKT/GSK3β, while LY294002 reversed the neuroprotective effect of OCN on PC12 cells (G) through inhibiting the phosphorylation of AKT and GSK3β (H) . Values were obtained from three independent experiments. * P < 0.05 (compared with the control group), # P < 0.05 (compared with the 6-OHDA group).

Article Snippet: A rat OCN ELISA kit was purchased from MyBioSource (San Diego, CA, USA, Catalog: MBS2022619).

Techniques: Flow Cytometry, CCK-8 Assay, Phospho-proteomics, Control

Journal: Open Biology

Article Title: Osteocyte TSC1 promotes sclerostin secretion to restrain osteogenesis in mice

doi: 10.1098/rsob.180262

Figure Lengend Snippet: Deletion of TSC1 in osteocytes stimulates osteogenesis and bone formation in mice. ( a ) Micro-CT reconstruction of distal femora from 4-, 8- and 12-week-old control (DTCL) and Tsc1 CKO mice. The scale bar represents 1 mm ( n = 6). ( b ) BMD of trabecular bone in the distal femora from 4-, 8- and 12-week-old control (DTCL) and Tsc1 CKO mice ( t -test, 4 weeks: p = 0.0083, 8 weeks: p = 0.0257, 12 weeks: p = 0.0284). BMD, bone mineral density ( n = 6). ( c ) Immunohistochemical staining and quantification of DMP1 in decalcified sections of distal femora from 10-week-old control (DTCL) and Tsc1 CKO mice ( t -test, p = 0.0037). Numbers of DMP1 (N. DMP1 + ). The scale bars represent 50 µm ( n = 6). ( d ) mRNA expression of DMP1 in femora from DTCL and Tsc1 CKO mice ( t -test, p = 0.0002, n = 6). ( e ) Calcein double labelling of cortical bone in distal femora from 10-week-old control (DTCL) and Tsc1 CKO mice. The boxed area is magnified in the panel below. The scale bars represent 100 µm and 50 µm ( n = 6). ( f ) Mineral apposition rate (MAR) ( t -test, p = 0.0062). ( g ) Immunohistochemical staining and quantification analysis of OCN in distal femora from 10-week-old control and Tsc1 CKO mice ( t -test, p = 0.0008). Numbers of OCN (N. OCN + ). The scale bars represent 50 µm ( n = 6). ( h ) mRNA level of osteocalcin (OCN) in DTCL and Tsc1 CKO mice ( t -test, p = 0.0008). ( i ) Enzyme-linked immunosorbent assay (ELISA) detection of serum OCN levels in control (DTCL) and Tsc1 CKO mice ( t -test, p = 0.0009, 12-week-old males, n = 6). ( j ) ELISA detection of serum P1NP levels in control and Tsc1 CKO mice ( t -test, p = 0.001, 12-week-old mice, n = 6). ( k ) ELISA detection of serum CTX-1 levels in control and Tsc1 CKO mice ( t -test, p = 0.029, 12-week-old mice, n = 6). ( l ) Western blot analysis of the expression levels of OCN protein in trabecular bone lysates from control and Tsc1 CKO mice. All experiments were repeated independently three times. Data are represented as mean ± s.d., * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: The protein levels of P1NP and OCN released into the culture supernatants were measured using a mouse OCN enzyme-linked immunosorbent assay (ELISA) kit (MyBioSource, San Diego, CA, USA; MBS268470), mouse P1NP ELISA kit (Houston, TX, USA) and CTX-1 ELISA kit (CEA665Mu; Cloud-Clone Co., Ltd, Wuhan, China).

Techniques: Micro-CT, Immunohistochemical staining, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Environmental Health Perspectives

Article Title: Ambient PM 2.5 Exposure and Bone Homeostasis: Analysis of UK Biobank Data and Experimental Studies in Mice and in Vitro

doi: 10.1289/EHP11646

Figure Lengend Snippet: The effect of PM 2.5 on bone morphology and osteogenesis in vivo . Mice were exposed to FA or PM 2.5 for 6 months. (A) Representative coronal images of intact femur. Scale bar: 1 mm . Quantitative analyses of femur (B) length and (C) width. (D) Representative images of 3D reconstruction of ROI. Scale bar: 500 μ m . Micro-CT quantitative analyses of (E) cortical bone mineral density and (F) trabecular bone mineral density. (G) Representative images of OCN immumohistochemical staining. Red arrows indicate positive expression. Scale bar: 50 μ m . (H) Quantification of OCN positive area. (I) Quantitative analyses of OCN levels in serum. (J) Representative images of ALP immumohistochemical staining. Red arrows indicate positive expression. Scale bar: 50 μ m . (K) Quantification of ALP positive area. (L) Quantitative analysis of BALP levels in serum. (M) Expression of ALP in liver assessed by western blotting. (N) Quantitative analysis of ALP expressions in liver. Numeric data can be found in Table S11. Data are presented as means ± SEMs (unpaired Student’s t -test, n = 4 – 6 ), * p < 0.05 , *** p < 0.001 . Note: 2D, two dimensions; 3D, three dimensions; ALP, alkaline phosphatase; BALP, bone alkaline phosphatase; BMD, bone mineral density; FA, filtered air; micro-CT, micro-computed tomography; OCN, osteocalcin; PM, particulate matter; PM 2.5 , particulate matter with aerodynamic diameter of ≤ 2.5 μ m ; ROI, region of interest; SEM, standard error of the mean.

Article Snippet: The enzyme-linked immunosorbent assay (ELISA) kit for bone alkaline phosphatase (BALP; #KE1424), osteocalcin (OCN; #KE1428), and TRAP 5b (TRACP-5b; #KE1580) were obtained from ImmunoWay Biotechnology, whereas the ELISA kit for TNF- α (#MTA00B) was purchased from R&D.

Techniques: In Vivo, Micro-CT, Staining, Expressing, Western Blot

Journal: Environmental Health Perspectives

Article Title: Ambient PM 2.5 Exposure and Bone Homeostasis: Analysis of UK Biobank Data and Experimental Studies in Mice and in Vitro

doi: 10.1289/EHP11646

Figure Lengend Snippet: The effect of PM 2.5 on osteoclastogenesis and inflammation in vivo . Mice were exposed to FA or PM 2.5 for 6 months. (A) Representative images of RANKL immunohistochemical staining around the growth plate. Red arrows indicate positive expression. Scale bar: 100 μ m . (B) Quantification of RANKL-positive area around the growth plate. (C) Representative images of TRAP staining around the femoral distal growth plate. Images in the right panel are higher magnifications of images in the respective left panel. Scale bar: 200 μ m . (D) Quantitative analysis of TRAP-positive cells around the femoral distal growth plate. (E) Quantitative analysis of TRACP-5b levels in serum. (F) Representative images of ABH/OG staining around the femoral distal growth plate. Red arrows indicate growth plate destruction. Scale bar: 50 μ m . (G) Representative images of Ctsk immunohistochemical staining. Red arrows indicate positive expression. Scale bar: 50 μ m . (H) Quantification of the Ctsk-positive area. (I) Representative images of TNF- α immumohistochemical staining. Red arrows indicate positive expression. Scale bar: 50 μ m . (J) Quantification of TNF- α –positive area. (K) Representative images of IL- 1 β immumohistochemical staining. Red arrows indicate positive expression. Scale bar: 50 μ m . (L) Quantification of IL- 1 β –positive area. Numeric data can be found in Table S12. Data are presented as means ± SEMs (unpaired Student’s t -test, n = 4 – 6 ), * p < 0.05 , ** p < 0.01 , *** p < 0.001 . Note: ABH/OG, Alcian blue hematoxylin/orange G; Ctsk, cathepsin K; FA, filtered air; IL- 1 β , interleukin-1beta; PM, particulate matter; PM 2.5 , particulate matter with aerodynamic diameter of ≤ 2.5 μ m ; RANKL, receptor activator of nuclear factor-kappa ligand; SEM, standard error of the mean; TNF- α , tumor necrosis factor-alpha; TRACP-5b, tartrate-resistant acid phosphatase 5b; TRAP, tartrate-resistant acid phosphatase.

Article Snippet: The enzyme-linked immunosorbent assay (ELISA) kit for bone alkaline phosphatase (BALP; #KE1424), osteocalcin (OCN; #KE1428), and TRAP 5b (TRACP-5b; #KE1580) were obtained from ImmunoWay Biotechnology, whereas the ELISA kit for TNF- α (#MTA00B) was purchased from R&D.

Techniques: In Vivo, Immunohistochemical staining, Staining, Expressing

Journal: Environmental Health Perspectives

Article Title: Ambient PM 2.5 Exposure and Bone Homeostasis: Analysis of UK Biobank Data and Experimental Studies in Mice and in Vitro

doi: 10.1289/EHP11646

Figure Lengend Snippet: The effect of PM stimulation on osteoclastogenesis in vitro . Preosteoclasts were induced and treated with conditional medium from PM 2.5 -stimulated RAW264.7 macrophages and primary BMMs. (A) TNF- α release from RAW264.7 macrophages in the supernatant after 24-h incubation with PM 2.5 particles dosed at 0, 25, and 50 μ g / mL . (B) Relative mRNA expression levels of molecules related to osteoclastogenesis in BMMs after treatment with 5% conditioned medium collected from supernatant of RAW264.7 macrophages. (C) Representative images of TRAP-stained cells treated with 5% conditioned medium collected from supernatant of 0, 25, and 50 μ g / mL PM 2.5 particles–incubated RAW264.7 macrophages. Scale bar: 100 μ m . (D) Quantification of TRAP-positive multinucleated cells with more than three nuclei on each well. (E) TNF- α release from BMMs in the supernatant after 24-h incubation with PM 2.5 particles dosed at 0, 25, and 50 μ g / mL . (F) Relative mRNA expression levels of molecules related to osteoclastogenesis in BMMs after treatment with 20% conditioned medium collected from supernatant of BMMs induced by PM 2.5 particles. (G) Representative images of TRAP-stained cells treated with 20% conditioned medium collected from supernatant of 0, 25, and 50 μ g / mL PM 2.5 particles–incubated BMMs. Scale bar: 100 μ m . (H) Quantification of TRAP-positive multinucleated cells with more than three nuclei on each well. Numeric data can be found in Table S13. Data are presented as means ± SEMs ( n = 3 – 4 ) and assessed through one-way ANOVA. * p < 0.05 , ** p < 0.01 , *** p < 0.001 vs. the 0 - μ g / mL group. # p < 0.05 , ## p < 0.01 , ### p < 0.001 vs. the 25 - μ g / mL group. Note: ACP5 , acid phosphatase 5; ANOVA, analysis of variance; Atp6v0d2 , ATPase, H + transporting, lysosomal V0 subunit D2; BMMs, bone marrow macrophages; c-Fos , FBJ murine osteosarcoma viral oncogene homolog; Ctsk , cathepsin K; Dcstamp , dendritic cell-specific transmembrane protein; Nfatc1 , nuclear factor of activated T cells cytoplasmic 1; PM, particulate matter; PM 2.5 , particulate matter with aerodynamic diameter of ≤ 2.5 μ m ; RAW264.7, a cell line of murine macrophages; SEM, standard error of the mean; TNF-α, tumor necrosis factor-alpha; Traf6 , tumor necrosis factor receptor-associated factor 6; TRAP, tartrate-resistant acid phosphatase; UD, undetectable.

Article Snippet: The enzyme-linked immunosorbent assay (ELISA) kit for bone alkaline phosphatase (BALP; #KE1424), osteocalcin (OCN; #KE1428), and TRAP 5b (TRACP-5b; #KE1580) were obtained from ImmunoWay Biotechnology, whereas the ELISA kit for TNF- α (#MTA00B) was purchased from R&D.

Techniques: In Vitro, Incubation, Expressing, Staining

Journal: Environmental Health Perspectives

Article Title: Ambient PM 2.5 Exposure and Bone Homeostasis: Analysis of UK Biobank Data and Experimental Studies in Mice and in Vitro

doi: 10.1289/EHP11646

Figure Lengend Snippet: The effect of TNF- α inhibition on PM 2.5 -stimulated osteoclastogenesis. Osteoclasts treated with conditional medium from PM 2.5 -stimulated primary BMMs were incubated with TNF- α neutralizing antibody D2H4. (A) Representative images of TRAP-stained BMMs treated with 20% conditioned medium and additional TNF- α neutralizing antibody D2H4. Scale bar: 100 μ m . (B) Quantification of TRAP-positive multinucleated cells with more than three nuclei on each well. (C) Representative images of bone resorption after treatment with 50 μ g / mL PM 2.5 particles–incubated BMMs with or without D2H4. Scale bar: 50 μ m . (D) Quantification of bone resorbed area in plates coated with hydroxyapatite. (E) Relative mRNA expression levels of molecules related to osteoclastogenesis in BMMs after treatment with conditioned medium collected from supernatant of 50 μ g / mL PM 2.5 particles incubation or plus TNF- α neutralizing antibody D2H4. (F) Representative bands of Traf6, c-Fos, and GAPDH in BMMs. (G) Quantification of Traf6 and c-Fos protein expression in PM-induced BMMs with or without D2H4. (H) Schematic diagram depicting the effect of PM 2.5 in bone homeostasis based on our findings. Numeric data can be found in Table S14. Data are presented as means ± SEMs ( n = 3 – 4 ) and assessed through ANOVA. * p < 0.05 , ** p < 0.01 , *** p < 0.001 vs. the 0 - μ g / mL group. # p < 0.05 , ## p < 0.01 vs. the 25 - μ g / mL group. $ p < 0.05 , $$ p < 0.01 vs. the 50 - μ g / mL group. Note: ANOVA, one-way analysis of variance; Atp6v0d2 , ATPase, H + transporting, lysosomal V0 subunit D2; BMMs, bone marrow macrophages; c-Fos , FBJ murine osteosarcoma viral oncogene homolog; D2H4, TNF- α neutralizing antibody; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Nfatc1 , nuclear factor of activated T cells cytoplasmic 1; OCN, osteocalcin; PM, particulate matter; PM 2.5 , particulate matter with aerodynamic diameter of ≤ 2.5 μ m ; SEM, standard error of the mean; TNF- α , tumor necrosis factor-alpha; Traf6 , tumor necrosis factor receptor-associated factor 6; TRAP, tartrate-resistant acid phosphatase.

Article Snippet: The enzyme-linked immunosorbent assay (ELISA) kit for bone alkaline phosphatase (BALP; #KE1424), osteocalcin (OCN; #KE1428), and TRAP 5b (TRACP-5b; #KE1580) were obtained from ImmunoWay Biotechnology, whereas the ELISA kit for TNF- α (#MTA00B) was purchased from R&D.

Techniques: Inhibition, Incubation, Staining, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Haptoglobin Acts as a TLR4 Ligand to Suppress Osteoclastogenesis via the TLR4-IFN-β Axis.

doi: 10.4049/jimmunol.1800661

Figure Lengend Snippet: FIGURE 1. The bone phenotype of Hp-deficient mice. The femurs of 8-wk-old WT or Hp-deficient (Hp2/2) male mice were analyzed by mCT and histological sectioning (n = 8). (A) The images represent femurs of WT or Hp2/2 mice. (B) Reconstructed three-dimensional mCT images were analyzed to measure bone parameters, such as bone volume per tissue volume (BV/TV), bone surface per bone volume (BS/BV), trabecular bone pattern factor (Tb.Pf), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). (C) Reconstructed three-dimensional mCT images were analyzed to measure cortical bone parameters, such as cortical thickness (Ct.Th), Tt.Ar, Ct.Ar, and cortical area fraction (Ct.Ar/Tt.Ar). (D) Decalcified femur bones were subjected to microdissection and stained for TRAP. Nuclei were counterstained with methyl green. Oc.S/BS, N.Oc./BS, osteoblast surface per bone surface (Ob.S/BS), and number of osteoblasts per bone surface (N.Ob./BS) were examined by using the OsteoMeasure program (black arrow, osteoclast; arrow head, osteoblast). (E) Serum levels of OCN and P1NP of 8-wk-old wild-type (WT) or Hp-deficient (Hp2/2) male mice were analyzed by ELISA. *p , 0.05, **p , 0.01, ***p , 0.001 (paired t test, two-sided).

Article Snippet: To analyze the secreted Hp, osteocalcin (OCN), procollagen type 1 N-terminal propeptide (P1NP) and IFN-b levels, mouse serum, or culture supernatant medium were collected and assessed using IFN-b ELISA kit (PBL Assay Science), OCN ELISA kit (LSBio, Seattle), P1NP ELISA kit (IDS, Boldon, U.K.), or a Hp ELISA kit (Abnova, Taipei City, Taiwan) according to the manufacturer’s instructions.

Techniques: Laser Capture Microdissection, Staining, Enzyme-linked Immunosorbent Assay