biotinylated rat anti mouse f4 80  (Bio-Rad)

Journal: The Journal of Clinical Investigation

Article Title: Reduction of SPARC protects mice against NLRP3 inflammasome activation and obesity

doi: 10.1172/JCI169173

Figure Lengend Snippet: ( A and B ) Schematic and weight change of Con and Adip-KO mice with 21 weeks of HFD followed by 13 weeks of chow diet ( n = 5, 5, respectively). ( C ) Body composition analysis of female Con and Adip-KO mice before and after diet change from HFD to chow diet ( n = 4, 4, respectively). ( D and E ) q-PCR analysis of inflammatory gene ( D ) and components of inflammasome ( E ) in VAT macrophages (F4/80 + ) in obese mice switched to chow diet ( n = 4, 4, respectively). ( F ) Inflammasome activation after pretreatment of SPARC protein for 24 hours following ATP (5mM) treatment with or without LPS (1 μg/mL) measured by caspase-1 Western blot analysis in cell lysate (lower) and supernatant (upper). Error bars represent the mean ± SEM. 2-tailed unpaired t tests were performed for statistical analysis. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: For positive selection of macrophages, SVF from VAT was incubated with biotinylated anti-mouse F4/80 antibody (eBioscience, 13-4801-85) in isolation buffer (PBS, 0.1% BSA, 2mM EDTA, pH 7.4).

Techniques: Activation Assay, Western Blot

Journal: bioRxiv

Article Title: Macrophages sense ECM mechanics and growth factor availability through cytoskeletal remodeling to regulate their tissue repair program

doi: 10.1101/2023.06.28.545586

Figure Lengend Snippet: ECM deposition during tissue repair in vivo induces cytoskeletal remodeling and suppresses mechanosensitive gene expression in macrophages. (A and B) Representative microscopic images of H&E staining (A) and Masson’s trichrome staining (B) of lungs at 3, 6, 9, and 12 days after oropharyngeal PBS or bleomycin administration to mice (n = 3/group/time point). Scale bar = 100 μm. (C) Relative gene expression in whole lungs from PBS- or bleomycin-treated mice (n = 6-7/group/time point). Statistically significant differences between time points in the bleomycin-treated group are shown. (D) FIZZ1 and ARG1 protein expression in lung macrophages in PBS- or bleomycin-treated mice, analyzed by flow cytometry following the gating strategy in Fig. S5. The percentage of FIZZ1-positive and ARG1-positive cells are shown (n = 4-5/group/time point). Statistically significant differences between time points in the bleomycin-treated group are indicated. (E) Representative snapshots of 3D-reconstructed confocal images of lungs from bleomycin-treated mice (n = 3/time point). Surfaces of F4/80 + cells (magenta) and collagen I (green) are shown. Scale bar = 20 μm. (F) Quantification of the sphericity of F4/80+ cells in (E). Two-way ANOVA (C and D) and one-way ANOVA (F) are used for statistical analysis. *p < 0.05, ** p < 0.01, **** p < 0.0001. ns; not significant.

Article Snippet: Following the wash with wash buffer, tissues were reacted with rabbit anti-collagen I antibody (ThermoFisher Scientific, PA5-95137) at 1:200 and biotinylated anti-F4/80 antibody (clone BM8, eBioscience, 13-4801-85) at 1:200 in blocking buffer overnight at 4°C.

Techniques: In Vivo, Expressing, Staining, Flow Cytometry

Journal: PLoS ONE

Article Title: Oral Treatment with γ-Aminobutyric Acid Improves Glucose Tolerance and Insulin Sensitivity by Inhibiting Inflammation in High Fat Diet-Fed Mice

doi: 10.1371/journal.pone.0025338

Figure Lengend Snippet: Groups of C57BL/6 mice were fed with HFD and provided with plain water or water containing 2 mg/ml of GABA for 20 weeks. The mice were sacrificed and the amounts of VAT in individual mice were measured. One portion of the VAT was fixed with 4% paraformaldehyde for 24 hours and the VAT sections at 5 µm were stained with Toluidine blue O, followed by examination under a light microscope. The sizes of adipocytes in 5 sections of individual mice from each group (n = 8) were examined in a blinded fashion. Another portion of VAT was fixed with the Bouin buffer for 48 hours and the VAT sections were subjected to immunohistochemistry analysis of infiltrated macrophages using anti-F4/80 antibodies and DAB substrate (brown), and the percentages of macrophages in 400 nuclear cells from 5 sections of each mouse in individual groups of mice were quantified in a blinded manner. Data are representative images of the adipocytes, macrophages stained and expressed as mean ± SEM from each group of mice (n = 8 for immunohistological examination and n = 12 for measuring the amounts of VAT per group). *p< 0.05; ** p<0.01 vs. the GABA-fed mice.

Article Snippet: The sections were probed with biotinylated anti-F4/80 (PharMigen, San Diego, USA) overnight at 4°C.

Techniques: Staining, Light Microscopy, Immunohistochemistry