Journal: PLoS Genetics

Article Title: Congenital lipodystrophy induces severe osteosclerosis

doi: 10.1371/journal.pgen.1008244

Figure Lengend Snippet: A) Transplanted fat depots 3 months after surgery. B) Fat depot weight 3 months after transplantation. C) Serum leptin and adiponectin of FF mice 3 months after fat depot transplantation. WT and non-transplanted FF mice serve as control. D) μCT analysis of trabecular bone volume and bone mineral density of distal femurs of FF mice 3 months after sham operation or transplantation of fat derived from WT or adipokine-deficient mice. Data are presented as mean ± SD. **p<0.01; *** p<0.001 as determined by ANOVA with Holm-Sidak's post hoc analysis for multiple comparisons test. D) comparison with FF Sham except where detailed.

Article Snippet: The primary antibody cocktail contained rat anti-mouse CD45-BUV395 (BD Horizon, clone 30-F11, final dilution factor 1:200), rat anti-mouse TER-119-APC (BioLegend, clone TER-119, 1:200), rat anti-mouse CD41-BV421 (BioLegend, clone MWReg30, 1:300), rat anti-mouse/human CD11b (BioLegend, clone M1/70, 1:400), and rat-anti mouse Leptin receptor (LepR)-biotin (R&D Systems, polyclonal, 1:50) in Brilliant Stain Buffer (BD Biosciences) containing 10 μg/mL FcBlock.

Techniques: Transplantation Assay, Control, Derivative Assay, Comparison

Journal: Scientific Reports

Article Title: Involvement of leptin signaling in the development of cannabinoid CB2 receptor-dependent mirror image pain

doi: 10.1038/s41598-018-28507-6

Figure Lengend Snippet: Leptin receptor expression was upregulated in nerve-injured CB2-KO animals 14 days after the surgery. Immunostaining of sciatic nerves using leptin ( a ) and leptin receptor ( b ) specific antibodies. Shown are the areas just next to the injured site (ipsilateral) or middle of dissected nerve (contralateral). Bars indicate a length of 100 μm. Leptin receptor expression was robustly increased in injured nerve of CB2-KO mice and a slight upregulation of leptin receptor expression was also observed on the contralateral nerve in CB2-KOs, but not in WT animals. The immunoreactivity was quantified (1 section/animal, n = 5) and expressed as percentage of positive signals to the stained area (mean ± SEM). A significant genotype effect is indicated by *p < 0.05 or ***p < 0.001, compared with respective WT controls (two-way ANOVA followed by Sidak’s multiple comparisons test). ( c ) A prominent induction of Leptin receptor expression was also observed in dorsal root ganglia of CB2-KO mice ipsilateral to the nerve injury.

Article Snippet: The slides were incubated in solutions containing anti-leptin antibody (1:100; R&D Systems), anti-leptin receptor antibody (1:40; R&D Systems) or anti-F4/80 antibody (1:100; Cedarlane) at 4 °C overnight, followed by incubation with Alexa488 or CY3 anti-goat secondary antibody (1:200; Jackson Immunoresearch, for anti-leptin and anti-leptin receptor) and Alexa488 anti-rat secondary antibody (1:200; Molecular Probes, for anti-F4/80) for 2 h at room temperature.

Techniques: Expressing, Immunostaining, Staining

Journal: Scientific Reports

Article Title: Involvement of leptin signaling in the development of cannabinoid CB2 receptor-dependent mirror image pain

doi: 10.1038/s41598-018-28507-6

Figure Lengend Snippet: Leptin receptor upregulation on sciatic nerve depends on the days post-surgery. Left panels, WT mice; right panels, CB2 knockouts, 3, 7 and 14 days after the nerve injury stained with anti-leptin receptor antibody. Starting 7-days after the surgery, leptin receptor expression increased on the injured nerve of CB2 knockouts and was further increased in nerve-injured CB2 knockouts after 14 postoperative days. A slight upregulation of leptin receptors was also seen on the non-injured nerve of CB2 knockouts at this time point. Leptin receptor upregulation at 14 postoperative days was further observed in injured nerve of WT animals. Quantified immunoreactivity is expressed as percentage of positive signals to the stained area (mean ± SEM). A significant genotype effect is indicated by **p < 0.01 or ***p < 0.001, compared with respective 0-days controls (two-way ANOVA followed by Sidak’s multiple comparisons test), n = 5. Figure . Nerve injury-induced STAT3 phosphorylation in peripheral nerve was enhanced in CB2 deficient animals, as evaluated by STAT3 and pSTAT3 western blotting of nerve samples 14-days after the surgery. Quantified data were normalised to ß-actin and expressed as means ± SEM of 5 mice/group. Open column, WT animals; closed column, CB2 knockout animals. A significant surgery or genotype effect is indicated by *p < 0.05 or ***p < 0.001, compared with respective WT controls (two-way ANOVA followed by Sidak’s multiple comparisons test).

Article Snippet: The slides were incubated in solutions containing anti-leptin antibody (1:100; R&D Systems), anti-leptin receptor antibody (1:40; R&D Systems) or anti-F4/80 antibody (1:100; Cedarlane) at 4 °C overnight, followed by incubation with Alexa488 or CY3 anti-goat secondary antibody (1:200; Jackson Immunoresearch, for anti-leptin and anti-leptin receptor) and Alexa488 anti-rat secondary antibody (1:200; Molecular Probes, for anti-F4/80) for 2 h at room temperature.

Techniques: Staining, Expressing, Phospho-proteomics, Western Blot, Knock-Out

Journal: Scientific Reports

Article Title: Involvement of leptin signaling in the development of cannabinoid CB2 receptor-dependent mirror image pain

doi: 10.1038/s41598-018-28507-6

Figure Lengend Snippet: CB2-KO mice show enhanced nerve injury-induced macrophage recruitment in peripheral nerves. Shown are immunostaining nerve tissues with F4/80 ( a ) and leptin receptor ( b ) 14 days after the surgery. Bars indicate a length of 100 μm, except for bars on close-up image indicating a length of 50 μm. Merged image ( c ) show that leptin receptor signals overlap with F4/80 signals,indicating infiltrated macrophages were expressing leptin receptors in CB2-KO mice. Quantified immunoreactivity is expressed as percentage of positive signals to the stained area (mean ± SEM). A significant genotype effect is indicated by ***p < 0.001, compared with respective WT controls (two-way ANOVA followed by Sidak’s multiple comparisons test), n = 5.

Article Snippet: The slides were incubated in solutions containing anti-leptin antibody (1:100; R&D Systems), anti-leptin receptor antibody (1:40; R&D Systems) or anti-F4/80 antibody (1:100; Cedarlane) at 4 °C overnight, followed by incubation with Alexa488 or CY3 anti-goat secondary antibody (1:200; Jackson Immunoresearch, for anti-leptin and anti-leptin receptor) and Alexa488 anti-rat secondary antibody (1:200; Molecular Probes, for anti-F4/80) for 2 h at room temperature.

Techniques: Immunostaining, Expressing, Staining

Journal: Scientific Reports

Article Title: Involvement of leptin signaling in the development of cannabinoid CB2 receptor-dependent mirror image pain

doi: 10.1038/s41598-018-28507-6

Figure Lengend Snippet: Perineurally administered leptin-neutralizing antibodies (20 ng/mouse/day) inhibited nerve injury-induced macrophage infiltration. Macrophage infiltration ( a ) as well as leptin receptor expression ( b ) was determined by immunohistochemistry 14 days after the surgery (24 hours after the final drug treatment). Bars indicate a length of 50 μm. ( c ) Merged images (green: F4/80, red: leptin receptor, blue: DAPI) showed that most of infiltrated macrophages in WT animals did not express leptin receptors. Quantified immunoreactivity is expressed as percentage of positive signals to the stained area (mean ± SEM). A significant treatment effect is indicated by **p < 0.01 or ***p < 0.001, compared with respective contralateral immunoreactivity (two-way ANOVA followed by Sidak’s multiple comparisons test), n = 5.

Article Snippet: The slides were incubated in solutions containing anti-leptin antibody (1:100; R&D Systems), anti-leptin receptor antibody (1:40; R&D Systems) or anti-F4/80 antibody (1:100; Cedarlane) at 4 °C overnight, followed by incubation with Alexa488 or CY3 anti-goat secondary antibody (1:200; Jackson Immunoresearch, for anti-leptin and anti-leptin receptor) and Alexa488 anti-rat secondary antibody (1:200; Molecular Probes, for anti-F4/80) for 2 h at room temperature.

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Scientific Reports

Article Title: Involvement of leptin signaling in the development of cannabinoid CB2 receptor-dependent mirror image pain

doi: 10.1038/s41598-018-28507-6

Figure Lengend Snippet: Perineurally administered leptin neutralizing antibodies (20 ng/mouse/day) inhibited nerve injury-induced macrophage infiltration in CB2-KO animals. F4/80 ( a ) and leptin receptor expression ( b ) as determined by immunohistochemistry 14 days after the surgery (24 hours after the final drug treatment). Bars indicate a length of 50 μm. ( c ) Merged images (green: F4/80, red: leptin receptor, blue: DAPI) showed that most of infiltrated macrophages in CB2-KO animals were leptin receptor positive. Quantified immunoreactivity is expressed as percentage of positive signals to the stained area (mean ± SEM). A significant treatment effect is indicated by *** p < 0.001, compared with respective contralateral immunoreactivity (two-way ANOVA followed by Sidak’s multiple comparisons test), n = 5.

Article Snippet: The slides were incubated in solutions containing anti-leptin antibody (1:100; R&D Systems), anti-leptin receptor antibody (1:40; R&D Systems) or anti-F4/80 antibody (1:100; Cedarlane) at 4 °C overnight, followed by incubation with Alexa488 or CY3 anti-goat secondary antibody (1:200; Jackson Immunoresearch, for anti-leptin and anti-leptin receptor) and Alexa488 anti-rat secondary antibody (1:200; Molecular Probes, for anti-F4/80) for 2 h at room temperature.

Techniques: Expressing, Immunohistochemistry, Staining