Journal: Journal of Neuroinflammation

Article Title: Presence and activation of pro-inflammatory macrophages are associated with CRYAB expression in vitro and after peripheral nerve injury

doi: 10.1186/s12974-021-02108-z

Figure Lengend Snippet: Quantification of the number of pro-inflammatory and immunosuppressive macrophages in WT and Cryab −/− crushed sciatic nerves. Representative images and quantification of the number of CD16/32 + Iba1 + DAPI + ( a , c ) and CD206 + Iba1 + DAPI + ( b , d ) profiles in the sciatic nerves of naïve (N), and 3, 5, 7, 14, 21, and 28 days post-crushed WT (white bars) and Cryab −/− (black bars) animals; representative of 2 experiments, n =3–4 animals/group, bar = 20 μm, data represent mean ± sem, * p < 0.05 independent t -test. e Graph displaying the ratio of CD16/32 + cells to CD206 + cells for WT and Cryab − / − nerves

Article Snippet: The tissues were incubated overnight at 4 °C with rabbit anti-Iba1 (Wako Chemicals, 019-1974, 1:200) or goat anti-Iba1 (Novus Biologicals, NB100-1028, 1:100), rat anti-mouse CD16/32 (BD Biosciences, 553842, 1:500), goat anti-mouse CD206 (Santa Cruz Biotechnologies, sc-34577, 1:500), chicken anti-P0 (Abcam, ab39375, 1:500), rabbit anti-CD68 (Cell Signaling, 97778, 1:100), rabbit anti-arginase 1 (Cell Signaling, 93668, 1:50), rabbit anti-iNOS (BD Transduction Laboratories, 1:100; gift from Dr. Samuel David, McGill University), and DAPI (Invitrogen, D3571, 1:2000).

Techniques:

Journal: Stem cell research & therapy

Article Title: Implantation and tracing of green fluorescent protein-expressing adipose-derived stem cells in peri-implant capsular fibrosis.

doi: 10.1186/s13287-023-03248-0

Figure Lengend Snippet: Fig. 2 A, B Representative images and quantification of α-smooth muscle actin (αSMA) in the control and green fluorescent protein-adipose-derived stem cell (GFP-ASC) groups. C, D Representative images and quantification of F4/80-positive macrophages in the control and GFP-ASC groups. E, F Representative images and quantification of iNOS-positive cells in F4/80-positive macrophages in the control and GFP-ASC groups. G, H Representative images and quantification of CD206-positive cells in F4/80-positive macrophages in the control and GFP-ASC groups. Values are presented as mean ± SD (n = 6). Scale bars = 100 μm. **p < 0.01. ***p < 0.001

Article Snippet: The cryosectioned slides were blocked with 5% bovine serum albumin diluted in PBS for 1 h at 37 °C, and then incubated with one of the following primary antibodies overnight at 4 °C: rabbit anti-mouse GFP antibody (AB3080; Millipore, Darmstadt, Germany), goat anti-mouse GFP antibody (ab5450; Abcam, Cambridge, UK), rat anti-mouse Ki67 antibody (14-5698-82; Invitrogen, Waltham, USA), rabbit anti-mouse PH3 antibody (9664; Cell signaling, Danvers, USA), rabbit anti-mouse Caspase3 antibody (53348; Cell signaling, Danvers, USA), mouse Cy3-conjugated anti-α-smooth muscle actin (αSMA) antibody (clone 1A4; Merck, Kenilworth, USA), rat anti-mouse F4/80 antibody (MAB5580; R&D Systems, Minneapolis, USA), mouse anti-mouse inducible nitric oxide synthase (iNOS) antibody (MAB9502; R&D Systems, Minneapolis, USA), goat anti-mouse CD206 antibody (AF2535; R&D Systems, Minneapolis, USA), or an Armenian hamster anti-mouse CD31 antibody (clone 2H8; Millipore, Darmstadt, Germany).

Techniques: Control, Derivative Assay

Journal: PLoS ONE

Article Title: FITC Conjugation Markedly Enhances Hepatic Clearance of N-Formyl Peptides

doi: 10.1371/journal.pone.0160602

Figure Lengend Snippet: Liver sections were fixed with cold acetone for 10 min, washed in PBS and incubated overnight at 4°C with polyclonal rabbit anti-FPR1 (1:200), followed by Alexa Fluor ® -488 goat anti-rabbit (1:1000). Intense FPR1 staining can be observed along the sinusoids in mouse liver (A), while in human liver the staining was more evenly localized to both LSECs and Heps (B). Double staining was performed using polyclonal rabbit anti-FPR1, rat anti-mouse CD206, and goat anti-human CD206, followed by Alexa Fluor ® -488 and Alexa Fluor ® -594 secondary antibodies, respectively.

Article Snippet: Polyclonal rabbit anti-FPR1 (H-230-Santa Cruz Biotech), rat-anti mouse CD206, clone MCA2235 (AbD Serotec, Oxford, UK), goat anti-human CD206 (R&D Systems), Alexa Fluor ® -488 and -594 conjugated secondary antibodies (InVitrogen), and Draq5 (BioStatus Limited, Leicestershire, UK) were used for immunolabeling of cells and tissue.

Techniques: Incubation, Staining, Double Staining

Journal: Journal of Nanobiotechnology

Article Title: Exosomal Lnc NEAT1 from endothelial cells promote bone regeneration by regulating macrophage polarization via DDX3X/NLRP3 axis

doi: 10.1186/s12951-023-01855-w

Figure Lengend Snippet: HUVECs-derived Exos attenuated inflammation by enhancing M2 polarization in vitro . A Representative images of the percentage of CD86 and CD206 positive cells detected by flow cytometry analysis. B Quantification of flow cytometry analysis of the percentage of CD86 and CD206 positive cells. C The concentrations of IL-1β, IL-6 and IL-10 of the supernatants detected by ELISA. D The relative gene expression of IL-1β, IL-6, IL-10 and Arg1 detected by qRT-PCR. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For immunofluorescence staining, to detect the macrophages, the sections were incubated with primary antibodies against CD86 (1:100, A2353, Abclonal) and CD206 (1:2000, 60143-1-IG, PTG) overnight at 4 °C, followed with secondary antibodies (Goat Anti-Rabbit IgG H&L (HRP), ab205718, Abcam, Goat Anti-Mouse IgG H&L (HRP), ab6789, Abcam) at room temperature for 1 h in the dark.

Techniques: Derivative Assay, In Vitro, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR

Journal: Journal of Nanobiotechnology

Article Title: Exosomal Lnc NEAT1 from endothelial cells promote bone regeneration by regulating macrophage polarization via DDX3X/NLRP3 axis

doi: 10.1186/s12951-023-01855-w

Figure Lengend Snippet: Air pouch model in vivo . A Schematic illustration of air pouch model establishment. B ELISA analysis of IL-1β, IL-6 and IL-10 concentration of lavage fluid. C Representative confocal images for CD86 (green) and CD206 (red). The nucleus were counterstained with DAPI (blue). D Quantification of the ratio of CD206/CD86 positive cells per field. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For immunofluorescence staining, to detect the macrophages, the sections were incubated with primary antibodies against CD86 (1:100, A2353, Abclonal) and CD206 (1:2000, 60143-1-IG, PTG) overnight at 4 °C, followed with secondary antibodies (Goat Anti-Rabbit IgG H&L (HRP), ab205718, Abcam, Goat Anti-Mouse IgG H&L (HRP), ab6789, Abcam) at room temperature for 1 h in the dark.

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Journal of Nanobiotechnology

Article Title: Exosomal Lnc NEAT1 from endothelial cells promote bone regeneration by regulating macrophage polarization via DDX3X/NLRP3 axis

doi: 10.1186/s12951-023-01855-w

Figure Lengend Snippet: Immunohistochemistry and immunofluorescence staining of bone defect. Representative images of A CD31, ALP, OCN, RUNX2 and NLRP3. Quantitative analysis of B CD31, C ALP, D OCN, E RUNX2 and F NLRP3. G Representative confocal images of calvarial sections for CD86 (green) and CD206 (red). In all images the nucleus were counterstained with DAPI (blue). H Quantification of the ratio of CD206/CD86 positive cells per field. Scale bar = 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For immunofluorescence staining, to detect the macrophages, the sections were incubated with primary antibodies against CD86 (1:100, A2353, Abclonal) and CD206 (1:2000, 60143-1-IG, PTG) overnight at 4 °C, followed with secondary antibodies (Goat Anti-Rabbit IgG H&L (HRP), ab205718, Abcam, Goat Anti-Mouse IgG H&L (HRP), ab6789, Abcam) at room temperature for 1 h in the dark.

Techniques: Immunohistochemistry, Immunofluorescence, Staining

Journal: Journal of Neuroinflammation

Article Title: Heterogeneous induction of microglia M2a phenotype by central administration of interleukin-4

doi: 10.1186/s12974-014-0211-6

Figure Lengend Snippet: Gene expression of M2a, M2c, and M1 polarization markers. Expression of CD206 (A) , TGFβ (B) , IL1β (C) and TNFα (D) genes was analyzed by real time PCR on RNA extracted from the frontal cortex (Fr cortex) and striatum of mice treated by intracerebroventricular injection for 16 and 48 h with saline (veh, open bars) or IL4 (black bars), as indicated. Data sets for each gene were calculated with the 2 -ddCt method with respect to the mean value of each vehicle group. Bars represent mean values ± SEM. * P < 0.05 versus veh 16 h; bb P < 0.05 versus striatum 16 h ( n = 4 to 6).

Article Snippet: Blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:2000, for Ym1 detection) or horse anti-goat IgG (1:2000, for CD206 detection; both from Vector Laboratories, Peterborough, UK) in incubation solution, for 1 h at room temperature.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Injection

Journal: Journal of Neuroinflammation

Article Title: Heterogeneous induction of microglia M2a phenotype by central administration of interleukin-4

doi: 10.1186/s12974-014-0211-6

Figure Lengend Snippet: Time course of IL4-induced M2a protein expression in mouse brain. After intracerebroventricular injection of saline (Veh, open bars) or IL4 (black bars), the striatum and frontal cortex (Fr. cortex) were extracted at 16 and 48 h intervals and analyzed by Western blotting to evaluate Ym1 (A , B) and CD206 (C , D) protein levels. (A , C) Immunoblots using Ym1, CD206, and β-actin antibodies from representative samples are shown. The average densitometric value of the Ym1 (B) and CD206 (D) bands from Western blots of several samples was normalized to that of β-actin; bars represent mean values ± SEM; the arbitrary value of 1 was given to the value of the saline 16 h group for each protein and brain area analyzed ( n = 4 to 6). BMDM, bone-marrow-derived macrophages.

Article Snippet: Blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:2000, for Ym1 detection) or horse anti-goat IgG (1:2000, for CD206 detection; both from Vector Laboratories, Peterborough, UK) in incubation solution, for 1 h at room temperature.

Techniques: Expressing, Injection, Western Blot, Derivative Assay