goat anti canine ccl2 igg  (R&D Systems)

Journal: Disease Models & Mechanisms

Article Title: Serum inflammatory cytokines as disease biomarkers in the DE50-MD dog model of Duchenne muscular dystrophy

doi: 10.1242/dmm.049394

Figure Lengend Snippet: Serum inflammatory cytokine concentrations in DE50-MD and WT dogs. Comparison of Luminex assay results for DE50-MD (grey) and WT (white) dog serum concentrations for (A) CCL2, (B) GM-CSF, (C) KC-like protein, (D) TNFα, (E) IFNγ and (F) IP-10. Dogs were studied longitudinally between 3 and 18 months of age. Month 3: WT, n =11; DE50-MD, n =12. Month 6: WT, n =11; DE50-MD, n =12. Month 9: WT, n =11; DE50-MD, n =8. Month 12: WT, n =11; DE50-MD, n =7. Month 15: WT, n =9; DE50-MD, n =6. Month 18: WT, n =9; DE50-MD, n =5. Boxes extend from the 25th to 75th percentile, with a line within the box at the median value. Each point represents an individual dog, and whiskers show the minimum and maximum results for that age group. Results that were below the lower limit of detection of the assay are presented as the minimum detectable concentration. Concentrations varied over orders of magnitudes between and/or within groups for some of the cytokines measured; therefore, a logarithmic scale was used on the y -axis to better display the data for graphs A,B,D and F. Asterisks indicate significance level based on linear mixed-model analysis, adjusted for repeated measures. * P <0.05; ** P <0.01; *** P <0.001; **** P <0.0001.

Article Snippet: Sections were then incubated with the following combinations of primary antibodies for 1 h at room temperature: goat anti-canine CCL2 IgG (R&D systems, AF1774, 1:20) with rat anti-human perlecan (Millipore, clone A7L6, 1:1000) , mouse anti-canine CD18 (Bio-Rad, monoclonal IgG1, clone CA1.4E9, MA1-82363, 1:100) ( C) or mouse anti-human macrophages (Biorad, monoclonal IgG1, clone MAC387, MCA874GT, 1:100) ( D).

Techniques: Comparison, Luminex, Concentration Assay

Journal: Disease Models & Mechanisms

Article Title: Serum inflammatory cytokines as disease biomarkers in the DE50-MD dog model of Duchenne muscular dystrophy

doi: 10.1242/dmm.049394

Figure Lengend Snippet: RT-qPCR of CCL2 mRNA in vastus lateralis muscle of DE50-MD and WT dogs. Results were normalised to the expression of three reference genes: RPL13A , HPRT1 and SDHA . Boxes extend from the 25th to 75th percentile, with a line within the box at the median value. Each point represents an individual DE50-MD or WT dog studied longitudinally between 3 and 18 months of age, and whiskers show the minimum and maximum results for that age group. Month 3: WT, n =4; DE50-MD, n =9. Month 6: WT, n =10; DE50-MD, n =10. Month 9: WT, n =10; DE50-MD, n =7. Month 12: WT, n =9; DE50-MD, n =6. Month 15: WT, n =8; DE50-MD, n =5. Month 18: WT, n =9; DE50-MD, n =6. Asterisks denote the level of significance of a difference between genotypes based on linear mixed-model analysis, adjusted for repeated measures. * P <0.05; ** P <0.01.

Article Snippet: Sections were then incubated with the following combinations of primary antibodies for 1 h at room temperature: goat anti-canine CCL2 IgG (R&D systems, AF1774, 1:20) with rat anti-human perlecan (Millipore, clone A7L6, 1:1000) , mouse anti-canine CD18 (Bio-Rad, monoclonal IgG1, clone CA1.4E9, MA1-82363, 1:100) ( C) or mouse anti-human macrophages (Biorad, monoclonal IgG1, clone MAC387, MCA874GT, 1:100) ( D).

Techniques: Quantitative RT-PCR, Expressing

Journal: Disease Models & Mechanisms

Article Title: Serum inflammatory cytokines as disease biomarkers in the DE50-MD dog model of Duchenne muscular dystrophy

doi: 10.1242/dmm.049394

Figure Lengend Snippet: Relationship between serum CCL2 protein and vastus lateralis muscle CCL2 mRNA. Graph relationships between the relative levels of CCL2 mRNA in the vastus lateralis muscle and serum CCL2 protein concentration (A), and between the relative CCL2 and CCR2 mRNA levels in the vastus lateralis muscle (B) of DE50-MD (black triangles) and WT (open circles). No relationship was found between serum CCL2 protein concentration and vastus lateralis muscle CCL2 mRNA levels within genotypes (DE50-MD: P =0.59, n =36 samples from a total of 13 dogs; WT: P =0.95, n =45 samples from a total of 11 dogs). A positive association between CCL2 and CCR2 mRNA levels in the vastus lateralis muscle was found for both WT ( P <0.0001, n =49 samples from a total of 11 dogs) and DE50-MD ( P =0.001, n =42 samples from a total of 13 dogs) subjects, based on linear mixed-model analysis. Repeated measures were accounted for in the statistical analyses.

Article Snippet: Sections were then incubated with the following combinations of primary antibodies for 1 h at room temperature: goat anti-canine CCL2 IgG (R&D systems, AF1774, 1:20) with rat anti-human perlecan (Millipore, clone A7L6, 1:1000) , mouse anti-canine CD18 (Bio-Rad, monoclonal IgG1, clone CA1.4E9, MA1-82363, 1:100) ( C) or mouse anti-human macrophages (Biorad, monoclonal IgG1, clone MAC387, MCA874GT, 1:100) ( D).

Techniques: Protein Concentration

Journal: Disease Models & Mechanisms

Article Title: Serum inflammatory cytokines as disease biomarkers in the DE50-MD dog model of Duchenne muscular dystrophy

doi: 10.1242/dmm.049394

Figure Lengend Snippet: Relationship between serum CCL2 protein concentration and other biomarkers of disease in DE50-MD dogs. (A) Graph relationship between serum CCL2 concentration and plasma CK activity. A positive relationship was found between the two variables (R 2 =0.37, P <0.0001). n =41 samples, from a total of 14 DE50-MD dogs studied longitudinally between 3 and 18 months of age (month 3, n =11; month 6, n =9; month 9, n =7; month 12, n =5; month 15, n =5; month 18, n =4). (B) Graph relationship between serum CCL2 concentration and the geometric mean (geomean) of acid phosphatase-stained fraction of vastus lateralis muscle biopsy samples. A positive relationship was found between the two variables in DE50-MD dogs (R 2 =0.41, P =0.0008). n =42 samples, from a total of 14 DE50-MD dogs studied longitudinally between 3 and 18 months of age (month 3, n =7; month 6, n =10; month 9, n =8; month 12, n =7; month 15, n =5; month 18, n =5). Linear regressions were calculated based on linear mixed-model analysis, accounting for repeated measures.

Article Snippet: Sections were then incubated with the following combinations of primary antibodies for 1 h at room temperature: goat anti-canine CCL2 IgG (R&D systems, AF1774, 1:20) with rat anti-human perlecan (Millipore, clone A7L6, 1:1000) , mouse anti-canine CD18 (Bio-Rad, monoclonal IgG1, clone CA1.4E9, MA1-82363, 1:100) ( C) or mouse anti-human macrophages (Biorad, monoclonal IgG1, clone MAC387, MCA874GT, 1:100) ( D).

Techniques: Protein Concentration, Concentration Assay, Clinical Proteomics, Activity Assay, Staining

Journal: Disease Models & Mechanisms

Article Title: Serum inflammatory cytokines as disease biomarkers in the DE50-MD dog model of Duchenne muscular dystrophy

doi: 10.1242/dmm.049394

Figure Lengend Snippet: Histology of the vastus lateralis muscle from WT and DE50-MD littermates from 3 to 18 months of age. (A,B) Immunohistochemistry (IHC) of vastus lateralis muscle from (A) a WT dog (ID WT-T3) and (B) a DE50-MD dog (ID DE50-T6). Each section was labelled with antibodies against perlecan (green) and CCL2 (magenta), and nuclei were labelled with Hoechst 33342 (blue). (C,D) For the DE50-MD dog DE50-T6, serial sections for each timepoint were stained for (C) acid phosphatase (AP) and with (D) H&E. Scale bar: 100 µm.

Article Snippet: Sections were then incubated with the following combinations of primary antibodies for 1 h at room temperature: goat anti-canine CCL2 IgG (R&D systems, AF1774, 1:20) with rat anti-human perlecan (Millipore, clone A7L6, 1:1000) , mouse anti-canine CD18 (Bio-Rad, monoclonal IgG1, clone CA1.4E9, MA1-82363, 1:100) ( C) or mouse anti-human macrophages (Biorad, monoclonal IgG1, clone MAC387, MCA874GT, 1:100) ( D).

Techniques: Immunohistochemistry, Staining

Journal: Disease Models & Mechanisms

Article Title: Serum inflammatory cytokines as disease biomarkers in the DE50-MD dog model of Duchenne muscular dystrophy

doi: 10.1242/dmm.049394

Figure Lengend Snippet: Histology of the vastus lateralis muscle from a 6-month-old DE50-MD dog. (A-E) Immunohistochemistry of a section of vastus lateralis muscle showing (A) CCL2 (magenta), (B) CD18 (green) and (C) a merged image of CCL2, CD18 and Hoechst 33342 (blue), with magnified views of the boxed region in C shown for (D) CCL2 and (E) CD18 staining. (F-J) Immunohistochemistry of a serial section showing (F) CCL2 (magenta), (G) MAC387 + regions (green) and (H) a merged image of CCL2, MAC387 and Hoechst 33342 (blue), with magnified views of the boxed region in H shown for (I) CCL2 and (J) CD18 staining. (K) A serial section with H&E staining showing cell infiltrate within and between muscle fibres, fibres with internalised nuclei and hypercontracted fibres (darker magenta). (L) Serial section with acid phosphatase (AP) staining showing positive red staining at areas of cell infiltrate and within some muscle fibres. (M-O) Serial section of immunohistochemistry with no primary antibody as a control with magnified views of the region of inflammation (dashed box) shown for the (N) magenta (CCL2) and (O) green (CD18/MAC387) channels. Scale bar: 100 µm.

Article Snippet: Sections were then incubated with the following combinations of primary antibodies for 1 h at room temperature: goat anti-canine CCL2 IgG (R&D systems, AF1774, 1:20) with rat anti-human perlecan (Millipore, clone A7L6, 1:1000) , mouse anti-canine CD18 (Bio-Rad, monoclonal IgG1, clone CA1.4E9, MA1-82363, 1:100) ( C) or mouse anti-human macrophages (Biorad, monoclonal IgG1, clone MAC387, MCA874GT, 1:100) ( D).

Techniques: Immunohistochemistry, Staining, Control

Journal: Disease Models & Mechanisms

Article Title: Serum inflammatory cytokines as disease biomarkers in the DE50-MD dog model of Duchenne muscular dystrophy

doi: 10.1242/dmm.049394

Figure Lengend Snippet: Samples tested for each study dog. (A) Serum samples tested by Luminex assay and (B) vastus lateralis muscle samples assayed by RT-qPCR for quantification of CCL2 and CCR2 mRNA. Age is displayed in months. Dog ID shows the study name of each individual animal included in this study: dog IDs with the prefix ‘DE50-‘ are DE50-MD dogs, dogs with the prefix ‘WT’ are wild-type dogs. White boxes show samples that were tested. Grey boxes indicate untested samples: dark grey boxes indicate samples that were unavailable due to animal euthanasia prior to this timepoint, whereas light grey boxes indicate samples unavailable for other reasons.

Article Snippet: Sections were then incubated with the following combinations of primary antibodies for 1 h at room temperature: goat anti-canine CCL2 IgG (R&D systems, AF1774, 1:20) with rat anti-human perlecan (Millipore, clone A7L6, 1:1000) , mouse anti-canine CD18 (Bio-Rad, monoclonal IgG1, clone CA1.4E9, MA1-82363, 1:100) ( C) or mouse anti-human macrophages (Biorad, monoclonal IgG1, clone MAC387, MCA874GT, 1:100) ( D).

Techniques: Luminex, Quantitative RT-PCR