nfix labelling  (Novus Biologicals)

Journal: bioRxiv

Article Title: Selective ablation of Nfix in Macrophages preserves Muscular Dystrophy by inhibiting FAPs-dependent fibrosis

doi: 10.1101/2021.05.12.443809

Figure Lengend Snippet: a) Percentage of F4/80 + MPs positive for Nfix in Tibialis Anterior muscles (TA) and diaphragm (Dia) of Sgca null mice at 2 and 6 months of life. b) Percentage of F4/80 + MPs positive for Nfix in Tibialis Anterior muscles (TA) and diaphragm (Dia) of mdx mice at 2 and 6 months of life. c) Percentage of Ly6C + and Ly6C - sorted MPs positive for Nfix from TA of Sgca null mice at 2, 3 and 4 months of life. Statistical significance was determined using a two-tailed Student’s t -test, one-way Anova test or two-way Anova test. * p<0,05; ** p<0,01 ; *** p<0,001 ; for c) * p<0,05 Ly6C - 2m vs 4m. Results are means ± SEM of at least three independent experiments.

Article Snippet: For Nfix-F4/80 double immunolabeling, cryosections were labelled with antibodies against F4/80 (1:400, Novus Biologicals NB300-605) overnight at 4°C and Nfix labelling using (1:200, Novus Biologicals NBP2-15038) antibody was performed for 2 hr at 37°C.

Techniques: Muscles, Two Tailed Test

Journal: bioRxiv

Article Title: Selective ablation of Nfix in Macrophages preserves Muscular Dystrophy by inhibiting FAPs-dependent fibrosis

doi: 10.1101/2021.05.12.443809

Figure Lengend Snippet: a) TGFβ1 expression by RT-qPCR of Nfix fl/fl :α (-/-) and ΦNfix (-/-) :α (-/-) TA muscles at 2, 4 and 6 months of life. b) Nfix fl/fl :α (-/-) and ΦNfix (-/-) :α (-/-) TA muscles were digested using Collagenase B and CD45 - cells were selected with magnetic beads. CD45 - cells were incubated with CD31-FITC, CD45-FITC, ITAG7(a7)-APC and Sca1-PeCy7 antibodies. FAPs are CD31 - /CD45 - /a7 - /Sca1 + . c) Nfix fl/fl :α (-/-) and ΦNfix (-/-) :α (-/-) TA muscles were digested using Collagenase B and CD45 + cells were selected with magnetic beads. CD45 + cells were incubated with CD64-APC and Ly6C-PE. MPs are CD64 + cells. Representative FACS gate of Ly6C + and Ly6C - MPs from Nfix fl/fl :α (-/-) and ΦNfix (-/-) :α (-/-) TA muscles at 2 months of life. d) Number of FAPs and MPs in Nfix fl/fl :α (-/-) and ΦNfix (-/-) :α (-/-) TA muscles at 2 and 4 months of life. e) Percentage of Ly6C + and Ly6C - MPs in Nfix fl/fl :α (-/-) and ΦNfix (-/-) :α (-/-) TA muscles at 2 and 4 months of life. Statistical significance was determined using a two-tailed Student’s t -test, one-way Anova test or two-way Anova test.* p<0,05 ; *** p<0,001. Results are means ± SEM of at least three independent experiments.

Article Snippet: For Nfix-F4/80 double immunolabeling, cryosections were labelled with antibodies against F4/80 (1:400, Novus Biologicals NB300-605) overnight at 4°C and Nfix labelling using (1:200, Novus Biologicals NBP2-15038) antibody was performed for 2 hr at 37°C.

Techniques: Expressing, Quantitative RT-PCR, Muscles, Magnetic Beads, Incubation, Two Tailed Test

Journal: bioRxiv

Article Title: Selective ablation of Nfix in Macrophages preserves Muscular Dystrophy by inhibiting FAPs-dependent fibrosis

doi: 10.1101/2021.05.12.443809

Figure Lengend Snippet: a) Immunostaining for PDGFRα (green) and Hoechst (blue) of Nfix fl/fl :α (-/-) and ΦNfix (-/-) :α (-/-) TA muscles at 2 months of life, and number of PDGFRα + cells by mm 2 . Immunostaining for F4/80 (green) and Hoechst (blue) of Nfix fl/fl :α (-/-) and ΦNfix (-/-) :α (-/-) TA muscles at 2 months of life, and number of F4/80 + cells by mm 2 . b) Western Blot of P-Smad3 and tot-Smad3 in Nfix fl/fl :α (-/-) and ΦNfix (-/-) :α (-/-) TA muscles at 2 months of life and quantification. Western blot were realized in double or membrane were stripped and vinculin was used to normalize. c) Strategy to sort MPs and FAPs by using CD45 magnetic beads on Nfix fl/fl :α (-/-) and ΦNfix (-/-) :α (-/-) hindlimb muscles at 2 and 4 months of life. d) Immunostaining for TNFα (red) or TGFβ (green) and Hoechst (blue) on cytospined sorted MPs and for aCaspase3 (red) or Ki67 (green) and Hoechst (blue) on cytospined sorted FAPs. e) Percentage of TNFα or TGFβ positive MPs and aCaspase3 or Ki67 positive FAPs sorted from Nfix fl/fl :α (-/-) and ΦNfix (-/-) :α (-/-) hindlimb muscles at 2 and 4 months of life. Statistical significance was determined using a two-tailed Student’s t -test or two-way Anova test. * p<0,05 ; ** p<0,01. Results are means ± SEM of at least three independent experiments. Scale bar = 50 μm for FAPs in a), and 100 μm for MPs in a), and b) and d).

Article Snippet: For Nfix-F4/80 double immunolabeling, cryosections were labelled with antibodies against F4/80 (1:400, Novus Biologicals NB300-605) overnight at 4°C and Nfix labelling using (1:200, Novus Biologicals NBP2-15038) antibody was performed for 2 hr at 37°C.

Techniques: Immunostaining, Muscles, Western Blot, Membrane, Magnetic Beads, Two Tailed Test

Journal: bioRxiv

Article Title: Selective ablation of Nfix in Macrophages preserves Muscular Dystrophy by inhibiting FAPs-dependent fibrosis

doi: 10.1101/2021.05.12.443809

Figure Lengend Snippet: a) 10 weeks old Sgca null and ΦNfix (-/-) :α (-/-) mice were i.p. injected 3 times per week for 2 weeks with physiological water or Y27632 ROCK inhibitor and analyzed. b) Number of F4/80 + cells per mm 2 of Sgca null and ΦNfix (-/-) :α (-/-) TA muscles of mice i.p. injected with physiological water or Y. c) Percentage of double Nfix + /Pax7 + cells and number of Pax7 + cells/mm 2 of Sgca null and ΦNfix (-/-) :α (-/-) TA muscles of mice i.p. injected with physiological water or Y. d) Percentage of Nfix + myofibers, CSA and percentage of perinucleated and centronucleated (from 1 to 3 + nuclei) myofibers of Sgca null and ΦNfix (-/-) :α (-/-) TA muscles of mice i.p. injected with physiological water or Y. Statistical significance was determined using one-way Anova test or two-way Anova test.* vs Sgca null: * p<0,05 ; ** p<0,01. $ vs Sgca null + Y: $ p<0,05. Results are means ± SEM of at least three independent experiments. Scale bar = 50μm.

Article Snippet: For Nfix-F4/80 double immunolabeling, cryosections were labelled with antibodies against F4/80 (1:400, Novus Biologicals NB300-605) overnight at 4°C and Nfix labelling using (1:200, Novus Biologicals NBP2-15038) antibody was performed for 2 hr at 37°C.

Techniques: Injection, Muscles

Journal: bioRxiv

Article Title: Selective ablation of Nfix in Macrophages preserves Muscular Dystrophy by inhibiting FAPs-dependent fibrosis

doi: 10.1101/2021.05.12.443809

Figure Lengend Snippet: a) Hematoxylin-eosin and Milligan Trichrome staining of Sgca null and ΦNfix (-/-) :α (-/-) TA muscles i.p. injected with physiological water or Y27632 (called Y). b) Immunostaining for Nfix (red), F4/80 (green) and Hoechst (blue) of Sgca null and ΦNfix (-/-) :α (-/-) TA muscles i.p. injected with physiological water or Y, and percentage of double Nfix + /F4/80 + cells. c) Immunostraining for Collagen I (green) of Sgca null and ΦNfix (-/-) :α (-/-) TA muscles i.p. injected with physiological water or Y, and percentage of Collagen I + area. d) Immunostaining for Lam (red), PDGFRα (green) and Hoechst (blue) of Sgca null and ΦNfix (-/-) :α (-/-) TA muscles i.p. injected with physiological water or Y, and number of PDGFRα + cells by mm 2 . Statistical significance was determined using a two-tailed Student’s t -test or one-way Anova test. * vs Sgca null: * p<0,05 ; ** p<0,01. $ vs Sgca null +Y: $$ p<0,01 ; $$$ p<0,001. Results are means ± SEM of at least three independent experiments. Scale bar = 50 μm.

Article Snippet: For Nfix-F4/80 double immunolabeling, cryosections were labelled with antibodies against F4/80 (1:400, Novus Biologicals NB300-605) overnight at 4°C and Nfix labelling using (1:200, Novus Biologicals NBP2-15038) antibody was performed for 2 hr at 37°C.

Techniques: Staining, Muscles, Injection, Immunostaining, Two Tailed Test