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

Journal: The Journal of Neuroscience

Article Title: Retinal Ganglion Cell Axon Regeneration Requires Complement and Myeloid Cell Activity within the Optic Nerve

doi: 10.1523/jneurosci.0555-21.2021

Figure Lengend Snippet: Figure 5. Marked increase in CR3 near the site of ONI. Complement C3b phagocytosis receptor CR3 is microglia/monocyte-specific, and CR31 microglia/monocyte number and phagocytic activation increase within the injured optic nerve. A, Representative images of CR3 immunolabeled uninjured (–) and injured (1, 3, 5, 7, 14 DPI) optic nerves at the injury site. Indicated regions (☐) are shown enlarged to the right of each image. Note the accumulation of CR31 cells and the increased cellular signal intensity following ONI. B, C, Quantitation confirms that the approximate number of CR31 cells (B) increases rapidly (by 1 DPI) and is sustained for at least 14 DPI, with a 30-fold peak at 5 DPI; likewise, CR3 immunofluorescence intensity (C) is elevated at all postinjury time points evaluated (1-14 DPI). D, The specificity of the CR3 (CD11b) antibody was verified using 14 DPI nerves from CR31/1 (left) and CR3/ mice (right). E, The approximate number of Iba11 microglia/monocytes in the nerve increases dramatically by 5 DPI and remains elevated until at least 14 DPI. F, Quantitation of CR31Iba1 (red), CR3-Iba11 (green), and colocalized CR31Iba11 (yellow) cells at/near the injury site. While most CR31 optic nerve cells are microglia, some CR31 cells in the injured nerve are likely infiltrating peripheral blood monocytes. G, CR3 (red) colocalization with Iba1 (green; microglia) at nerve injury site (3, 5 DPI), in the distal nerve (3 DPI), and in the chiasm (14 DPI). H–J, Presence of myeloid cell markers CD206 (H), CD45 (I), and P2RY12 (J) in 14 DPI optic nerves. Iba11 and CR31 microglia/monocytes at/near the injury are large, round, and lack processes: morphologic changes associated with phagocytic activation. L, Similarly, these Iba11 (green) microglia/monocytes express strikingly high levels of lysosome marker CD68 (red; observed 3-14 DPI, 14 DPI shown). M, C1q deletion dampens the CR31 cell response to injury plus zymosan 1 CPT-cAMP treatment at 5 and 14 DPI. N, Pro-regenerative treatments may augment the CR31 cell response in the injured nerve at 14 DPI. Scale bars: A, 50 mm, 10 mm; D, G, H, I, J, L, 50 mm. Graphs represent either mean 6 SEM or 25th to 75th percentile (box), median (line), range (whiskers), and individual data points (circles). B, C, E, K, *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001, one-way ANOVA with Dunnett post-test vs uninjured or untreated. J, **p , 0.01, t test.

Article Snippet: Antibodies used in immunohistochemistry experiments Antibody Target (specificity) Host species Manufacturer Dilution C1q Complement C1q (C1q) Rabbit Abcam #ab182451 1:250 C3 Complement C3 (C3) Goat MP/Cappel #55730 1:1000 C3d Complement C3d (C3d opsonin) Rabbit Dako #A0063 1:1000 CC1 CC1, APC (mature oligodendrocytes) Mouse Abcam #ab16794 1:100 CD11b Complement receptor 3, CR3, Mac1, integrin aMb 2 (monocytes, microglia, PMNs) Rat Bio-Rad #MCA711G 1:1000 CD45 PTPRC (microglia, monocytes, PMNs, NKs, lymphocytes) Rat Novus Biologicals #NB100-77417 1:500 CD68 Lysosomes (phagocytes) Rat Bio-Rad #MCA1957 1:200 CD206 Mannose receptor (monocytes, microglia) Goat R&D Systems #RB01 1:200 GAP43 Growth-associated protein 43 (regenerating axons) Sheep Custom Variable GFAP Glial fibrillary acidic protein (astrocytes) Rabbit Dako #Z0334 1:5000 GFAP Glial fibrillary acidic protein (astrocytes) Mouse Sigma #G3893 1:2500 Iba1 Ionized calcium binding adaptor molecule 1 (microglia, monocytes) Rabbit Wako #01919741 1:500 MBP Myelin basic protein (myelin) Rabbit Abcam #ab40390 1:500 Olig2 Oligodendrocyte transcription factor 2 (oligodendrocytes) Goat R&D Systems #AF2418 1:500 P2Y12 Purinergic receptor P2Y (microglia) Rabbit AnaSpec #AS-55043A 1:500 TUJ1 b -III tubulin (RGCs) Rabbit Abcam #ab18207 1:500 mean RGC count for each retina was transformed to RGC survival density (RGCs/mm2, divided by 0.235 mm2 image area), then expressed as %WT control.

Techniques: Activation Assay, Immunolabeling, Quantitation Assay, Immunofluorescence, Marker