Journal: Toxins

Article Title: Natriuretic-like Peptide Lebetin 2 Mediates M2 Macrophage Polarization in LPS-Activated RAW264.7 Cells in an IL-10-Dependent Manner

doi: 10.3390/toxins15040298

Figure Lengend Snippet: Effect of L2 and BNP on macrophage polarization in LPS-activated RAW264.7 cells. ( a ) Representative flow cytometry plots showing the variable macrophage distribution of M1-like macrophages (CD68 + /CD206 − cells, Q1 population) and M2-like macrophages (CD68 + /CD206 + /MRC-1, Q2 population) in control and treated cells. ( b , c ) Effect of L2 on M1 and M2 macrophage subtype expression. ( d , e ) Effect of BNP on M1 and M2 macrophage subtype expression. Cells were obtained after challenging RAW264.7 cells with LPS (1 µg/mL) for 24 h followed by 48 h treatment with or without L2 or BNP (0, 0.2, 0.4 and 0.8 ng/mL) and isatin (0.1 mM), added 20 min earlier. ( f ) M2/M1 ratio in LPS-activated RAW264.7 cells. The subtypes of macrophages were identified by analyzing profiles of cell surface markers by FACS. Pro-inflammatory M1 macrophages were identified as CD68 + /CD206 ࢤ cells and M2-like macrophages assessed by double immunolabeling of CD68 and CD206/MRC-1 in control and treated cells, and the data were analyzed by BD CellQuestPro software. All results were obtained from duplicate experiments. Data are reported as mean ± SEM. *** p < 0.001 vs. corresponding control LPS group; $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 vs. L2 corresponding group; †† p < 0.01 vs. group unstimulated with LPS.

Article Snippet: RAW264.7 macrophage polarization was detected after treatment with L2 and BNP by flow cytometric profiling of specific surface marker expression, including CD68 for total macrophage population quantification and CD206/MRC-1 for M2-like macrophage quantification using PE Rat Anti-Mouse CD68 and Alexa Fluor ® 647 Rat Anti-Mouse CD206 (BD BioScience, San Jose, CA, USA).

Techniques: Flow Cytometry, Expressing, Immunolabeling, Software

Journal: Toxins

Article Title: Natriuretic-like Peptide Lebetin 2 Mediates M2 Macrophage Polarization in LPS-Activated RAW264.7 Cells in an IL-10-Dependent Manner

doi: 10.3390/toxins15040298

Figure Lengend Snippet: Effect of L2 on macrophage polarization in LPS-activated RAW264.7 cells after interleukin-10 inhibition. ( a , b ) Effect of interlekin-10 (IL-10) on M1 and M2 macrophage subtype expression. Cells were obtained after challenging RAW264.7 cells with LPS for 24 h followed by 48 h treatment with or without exogenous IL-10 (0, 10 and 20 ng/mL). ( c , d ) Effect of L2 on M1 and M2 macrophage subtype expression after IL-10 inhibition. Cells were obtained after challenging RAW264.7 cells with LPS (1 µg/mL) for 24 h followed by 48 h treatment with or without L2 (0, 0.4 or 0.8 ng/mL) and IL-10 inhibitor at 10 µg/mL, added 20 min earlier. ( e ) Representative flow cytometry plots showing the variable macrophage distribution of M1-like macrophages (CD68 + /CD206 − cells, Q1 population) and M2-like macrophages (CD68 + /CD206 + /MRC-1, Q2 population) in control and treated cells. The subtypes of macrophages were identified by analyzing profiles of cell surface markers by FACS. Pro-inflammatory M1 macrophages were identified as CD68 + /CD206 − cells and M2-like macrophages assessed by double immunolabeling of CD68 and CD206/MRC-1 in control and treated cells, and the data were analyzed by BD CellQuestPro software. All results were obtained from duplicate experiments. Data are reported as mean ± SEM. *** p < 0.001 vs. corresponding control LPS group; $ p < 0.05, $$$ p < 0.001 vs. L2 corresponding group.

Article Snippet: RAW264.7 macrophage polarization was detected after treatment with L2 and BNP by flow cytometric profiling of specific surface marker expression, including CD68 for total macrophage population quantification and CD206/MRC-1 for M2-like macrophage quantification using PE Rat Anti-Mouse CD68 and Alexa Fluor ® 647 Rat Anti-Mouse CD206 (BD BioScience, San Jose, CA, USA).

Techniques: Inhibition, Expressing, Flow Cytometry, Immunolabeling, Software

Journal: Journal of Clinical Investigation

Article Title: B cells produce pathogenic antibodies and impair recovery after spinal cord injury in mice

doi: 10.1172/jci39780

Figure Lengend Snippet: Figure 4 Unilateral intraspinal microinjection of antibodies purified from SCI mice causes hind limb paralysis and neuropathology. (A and B) A sequence of still video images 1 day after injecting naive (uninjured) mice with control (uninjured) (A) or SCI antibodies (B). One complete step cycle is depicted in both cases. (C) Summary of hind limb function ipsilateral to the site of injection. Scoring is based on the BMS scale (0–5): 0, complete paralysis; 5, plantar stepping during more than 50% of step cycles. Scores above 5 were not considered, as our analyses were restricted to the limb on the injected side only. uninj, uninjured mice. **P < 0.01 versus uninjured; §P < 0.001 versus uninjured, 2-way ANOVA with Bonferroni’s post-hoc test. (D and E) Low- and high-power images from a mouse injected with control (D) or SCI antibodies (E), respectively. Note that intraspinal pathology is only evident in mice receiving SCI antibodies; the asterisk indicates the injection target. (F) Phagocytic microglia/ macrophages (red, anti-CD68) colocalize with axon/neuron pathology (green, anti–200-kDa NFH) at the site of injection in mice receiving SCI antibodies. (G–I) High-power images of boxed region in F. Scale bars: 0.2 mm (D–F); 50 μm (G–I).

Article Snippet: A list of primary antibodies and their final concentrations follows: rat antimouse C1q (0.133 μg/ml, clone 7H8; Abcam), rat anti-mouse CD45R/B220 (0.83 μg/ml, clone RA3-6B2; Serotec), mouse anti-mouse CD68 (1 μg/ml, clone FA-11; Serotec), chicken anti-mouse NFH (1 μg/ml; Aves Labs), mouse anti-MBP (1:40,000, clone SMI 94, mouse ascites; Covance), goat anti-mouse IgG (1 μg/ml, γ-chain specific; Southern Biotech), F(ab′)2 fragments of goat anti-mouse IgG heavy and light chains (1 μg/ml; Jackson ImmunoResearch Laboratories Inc.), and goat anti-mouse IgG F(ab′)2 fragments (1 μg/ml, γ-chain specific; Jackson ImmunoResearch Laboratories Inc.).

Techniques: Microinjection, Purification, Sequencing, Control, Injection

Journal: The Journal of Experimental Medicine

Article Title: Macrophages in the synovial lining niche initiate neutrophil recruitment and articular inflammation

doi: 10.1084/jem.20220595

Figure Lengend Snippet: Lining macrophages phagocytose ICs and model antigen-mBSA. (A) Quantification of PE fluorescence intensity by FACS in synovial resident macrophages 3 h after IA injections. Significance values by mixed-effects analysis with Šídák multiple comparisons post hoc: *, P < 0.05. GMOF, geometric mean of fluorescence. (B) Synovial neutrophil counts as a result of IC and PE challenge by FACS at 3 h after IA injections. Significance values by one-way ANOVA with Šídák multiple comparisons post hoc: *, P < 0.05; **, P < 0.01; data representative of two experiments. (C) Confocal fluorescence microscopy of the whole mouse knee in sagittal plane 4 h after injection of fluorescently labeled mBSA (large panel with white arrowheads pointing out lining and cartilage localization of the antigen, whereas red arrowhead indicates leak in the posterior part; pan-macrophage CD68, cyan; lining macrophage VSIG4, magenta; mBSA, yellow; nuclei, gray; colocalization of VSIG4 and mBSA, white; scale bar = 100 µm). (D) Zoomed region of the lining shows preferential uptake of mBSA by the lining macrophages and not the sublining ones (three right panels, scale bar = 20 µm). (E) Quantification of mBSA uptake by the lining and sublining macrophages ( N = 2; n = 6) in a scatter plot; each symbol indicates one section, and lines connect macrophages of the same section. N indicates the number of mice, n the number of quantified sections; data representative of two independent experiments. Significance values by Wilcoxon matched-pairs signed rank test: *, P < 0.05. FP, fat pad; M, meniscus; PT, patellar tendon; SC, synovial cavity.

Article Snippet: Total macrophages were labeled with either unconjugated rat anti-CD68 (Bio-Rad) and goat anti-rat Brilliant Violet 421 secondary antibody (BD Biosciences), or anti-CD68 directly conjugated to Brilliant Violet 421 (BD Biosciences), whereas goat-derived anti-VSIG4 (Bio-Techne) followed by donkey anti-goat Alexa Fluor 647 (Life Technologies) was used to delineate lining macrophages.

Techniques: Fluorescence, Microscopy, Injection, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Macrophages in the synovial lining niche initiate neutrophil recruitment and articular inflammation

doi: 10.1084/jem.20220595

Figure Lengend Snippet: Synovial lining macrophages can be identified and isolated by the expression of VSIG4. (A) Confocal image of the naive knee from CX3CR1-eGFP Ly6G-tdTomato double reporter mouse containing resident macrophages and no neutrophils; VISG4 and CX3CR1 colocalize in the lining (pan-macrophage CD68, cyan; lining macrophage VSIG4, magenta; CX3CR1, orange; nuclei, gray; sagittal plane; scale bar = 30 µm). (B) VISG4 marks lining macrophages by reanalysis of publicly available small bulk RNA sequencing dataset of murine paws in steady state . (C) Gating strategy used to isolate lining and sublining macrophages by FACS for small bulk RNA sequencing; pseudo-color plots of contralateral knee at 4 h p.c. created in FlowJo v10. F, femur; FP, fat pad; SC, synovial cavity; T, tibia.

Article Snippet: Total macrophages were labeled with either unconjugated rat anti-CD68 (Bio-Rad) and goat anti-rat Brilliant Violet 421 secondary antibody (BD Biosciences), or anti-CD68 directly conjugated to Brilliant Violet 421 (BD Biosciences), whereas goat-derived anti-VSIG4 (Bio-Techne) followed by donkey anti-goat Alexa Fluor 647 (Life Technologies) was used to delineate lining macrophages.

Techniques: Isolation, Expressing, RNA Sequencing Assay

Journal: The Journal of Experimental Medicine

Article Title: Macrophages in the synovial lining niche initiate neutrophil recruitment and articular inflammation

doi: 10.1084/jem.20220595

Figure Lengend Snippet: Activated transcriptional profile in lining macrophages at the onset of AIA and CXCL1 expression in the lining. (A) GO analysis of differentially expressed genes in lining and sublining macrophages at 4 h after mBSA challenge ( n = 3). FDR, false discovery rate. (B) GO analysis of differentially expressed genes in lining and sublining macrophages from PBS control knees ( n = 3). (C) Confocal images of the knee immunostained for CXCL1 at 4 h p.c. (large panel; VSIG4, magenta; CXCL1, green; CD68, cyan; CD31, red; nuclei, gray; scale bar = 100 µm). Zoomed region of the lining shows expression of CXCL1 in various cells (middle panel, scale bar = 10 µm), whereas further enlargement demonstrates colocalization of CXCL1 and VSIG4 (right panel, scale bar = 3 µm). (D) Quantification of CXCL1 distribution in the synovium in additional mice from two independent experiments; Each violin plot represents one to two knee sections of a single mouse with median and interquartile range indicated. F, femur; FP, fat pad; M, meniscus; T, tibia.

Article Snippet: Total macrophages were labeled with either unconjugated rat anti-CD68 (Bio-Rad) and goat anti-rat Brilliant Violet 421 secondary antibody (BD Biosciences), or anti-CD68 directly conjugated to Brilliant Violet 421 (BD Biosciences), whereas goat-derived anti-VSIG4 (Bio-Techne) followed by donkey anti-goat Alexa Fluor 647 (Life Technologies) was used to delineate lining macrophages.

Techniques: Expressing, Control

Journal: The Journal of Experimental Medicine

Article Title: Macrophages in the synovial lining niche initiate neutrophil recruitment and articular inflammation

doi: 10.1084/jem.20220595

Figure Lengend Snippet: Activation of lining macrophages at the onset of AIA. (A) Confocal fluorescence microscopy of mouse knee demonstrates CXCL1 immunostaining localizing in the lining at 4 h after AIA induction (large panel; VSIG4, magenta; CXCL1, green; CD68, blue; nuclei, gray; scale bar = 200 µm) with representative quantification of CXCL1 expression in regards to the synovial lining of the image in A; each point is one CXCL1 “surface” rendered by Imaris software with median and interquartile range indicated. Zoomed region of the lining shows expression of CXCL1 in the cells of the lining niche (bottom left panel, scale bar = 7 µm), whereas further enlargement demonstrates colocalization of CXCL1 and VSIG4 indicated with a white arrowhead, as well as CXCL1 expression in VSIG4 − lining cell indicated with a red arrowhead (bottom right panel, scale bar = 2 µm). F, femur; FP, fat pad; M, meniscus; PB, patellar bursa; T, tibia. Images representative of 10 sections from five different mice and two independent experiments. (B) Heatmap of genes within the GO term “Neutrophil recruitment” expressed in synovial macrophages shows higher expression of chemokines in lining cells; CXCL1 indicated with an arrowhead. (C) Confocal image of sub-patellar synovial region demonstrating fluorescent mBSA and CXCL1 coincidence in the lining area (left panel; VSIG4, magenta; CXCL1, green; CD68, blue; mBSA, yellow; nuclei, gray; scale bar = 40 µm). Enlarged region shows lining macrophages with intracellular mBSA and CXCL1 expression (right panel, arrowheads, scale bar = 7 µm). F, femur; P, patella; PB, patellar bursa; PT, patellar tendon.

Article Snippet: Total macrophages were labeled with either unconjugated rat anti-CD68 (Bio-Rad) and goat anti-rat Brilliant Violet 421 secondary antibody (BD Biosciences), or anti-CD68 directly conjugated to Brilliant Violet 421 (BD Biosciences), whereas goat-derived anti-VSIG4 (Bio-Techne) followed by donkey anti-goat Alexa Fluor 647 (Life Technologies) was used to delineate lining macrophages.

Techniques: Activation Assay, Fluorescence, Microscopy, Immunostaining, Expressing, Software

Journal: The Journal of Experimental Medicine

Article Title: Macrophages in the synovial lining niche initiate neutrophil recruitment and articular inflammation

doi: 10.1084/jem.20220595

Figure Lengend Snippet: Topography of recruited neutrophils during AIA: Onset-specific clustering in the synovial lining and interaction with mBSA-laden lining macrophages. (A and B) Confocal images of murine synovia during AIA in CX3CR1-eGFP Ly6G-tdTomato double reporter mice show that neutrophils cluster in the synovial lining niche at the onset: 6 h large overview (A) and 4 h anterior synovium ROI (B) have uniform distribution at day 2 (peak of inflammation, middle panel) and form localized swarms at day 7 (resolution of inflammation, bottom panel). CD68, cyan; CX3CR1, orange; VSIG4, magenta; Ly6G-tdTomato, green; nuclei, gray; scale bar = 300 and 50 µm in the entire mouse knee cross-section and ROIs, respectively. (C) Respective quantification of neutrophil position within the anterior synovium of the whole knee cross-sections as in A: neutrophil distance to the lining at the onset (6 h), peak (day 2), and resolution (day 7) of AIA presented in violin plots. Each violin plot represents three mice combined with two to three sections at different positions in the knee quantified, median and interquartile range indicated. Total quantifications: N = 3; n = 2–3; N indicates mouse group size, n the number of sections per mouse; samples from four independent experiments. P values by Kolmogorov–Smirnov comparison: ****, P < 0.0001. CL, cruciate ligaments; F, femur; FP, fat pad; M, meniscus; SC, synovial cavity. (D) Lining macrophages (L.MΦ) exhibiting higher mBSA uptake interact with more neutrophils (PMN) compared with the macrophages with lower mBSA uptake (whole anterior lining quantified on two different sections of a single mouse at 4 h p.c.; P value by Wilcoxon rank–sum test: *, P < 0.05). (E) Confocal image of the anterior synovium 4 h after injection of fluorescently labeled mBSA shows recruited neutrophils interacting with mBSA-laden lining macrophages (CD68, cyan; VSIG4, magenta; mBSA, yellow; neutrophil CD177, green; colocalization of VSIG4 and mBSA, white; scale bar = 10 µm). FP, fat pad; SC, synovial cavity.

Article Snippet: Total macrophages were labeled with either unconjugated rat anti-CD68 (Bio-Rad) and goat anti-rat Brilliant Violet 421 secondary antibody (BD Biosciences), or anti-CD68 directly conjugated to Brilliant Violet 421 (BD Biosciences), whereas goat-derived anti-VSIG4 (Bio-Techne) followed by donkey anti-goat Alexa Fluor 647 (Life Technologies) was used to delineate lining macrophages.

Techniques: Comparison, Injection, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Macrophages in the synovial lining niche initiate neutrophil recruitment and articular inflammation

doi: 10.1084/jem.20220595

Figure Lengend Snippet: Activation of microvasculature in the synovial lining niche at the onset of AIA. (A) Representative confocal image of the synovium demonstrating higher number of transferred CXCR2 OE T cells compared with the control T cells and the closer proximity of OE T cells to the lining (CD4 + ; CD8 + T cells, green; VSIG4, magenta; control T cells, cyan; CXCR2 OE T cells, orange; nuclei, gray; scale bar = 30 µm); single experiment with two animals. (B) Numbers ratio of CXCR2 OE T cells to control T cells measured by FACS in the blood and the synovium of mice 6 h p.c. (5 h after adoptive T cell transfer) shows preferential recruitment of CXCR2 OE T cells to the synovium. Significance value by paired t test: *, P < 0.05. Experiment performed two times. (C) CXCR2 OE T cells in the synovial cavity interacting with the lining macrophage (CD177, green; VSIG4, magenta; CD31, blue; control T cells, cyan; CXCR2 OE T cells, orange; nuclei, gray; scale bar = 10 µm). (D) Confocal image of the whole mouse knee cross-section immunostained for endothelial activation marker E-selectin (CD62E) reveals distinct position of activated vasculature close to the lining (CD62E, green; CD31, magenta; CD68, cyan; lining fibroblast Prg4, orange; nuclei, gray; scale bar = 200 µm). (E) Segmentation of the vasculature in the anterior synovium of the image in A using Imaris to classify highly activated (CD62E high ) and non-activated (CD62E −/low ) blood vessels. Inset shows representative distribution of activated microvasculature in regards to the synovial lining of the image mask in E; each point is one blood vessel surface of the respective image; median and interquartile range indicated. (F) Enlarged region of the synovial lining with CD62E high blood vessel is shown in the left panel, whereas the right panel shows patellar region with CD62E high blood vessels in direct contact with lining macrophages (arrowheads), as well as an activated larger-diameter blood vessel situated a few cell layers deeper (scale bar = 20 µm). B, bone; F, femur; FP, fat pad; M, meniscus; SC, synovial cavity; T, tibia. Images and quantification representative of four animals from two independent experiments.

Article Snippet: Total macrophages were labeled with either unconjugated rat anti-CD68 (Bio-Rad) and goat anti-rat Brilliant Violet 421 secondary antibody (BD Biosciences), or anti-CD68 directly conjugated to Brilliant Violet 421 (BD Biosciences), whereas goat-derived anti-VSIG4 (Bio-Techne) followed by donkey anti-goat Alexa Fluor 647 (Life Technologies) was used to delineate lining macrophages.

Techniques: Activation Assay, Control, Marker

Journal: The Journal of Experimental Medicine

Article Title: Macrophages in the synovial lining niche initiate neutrophil recruitment and articular inflammation

doi: 10.1084/jem.20220595

Figure Lengend Snippet: Visualizing activated endothelium and IRF5-dependent activation of the synovial lining. (A) Distance of activated and non-activated vasculature to the lining at 4 h p.c. in three different mice from two experiments. Each violin plot represents two to three knee sections of a single mouse with median and interquartile range indicated. P values by Wilcoxon rank–sum test: ****, P < 0.0001. (B) Staining of IRF5 demonstrates expression in VISG4 + lining macrophages that is higher compared to inflammatory monocytes and neutrophils by FACS. (C) Representative confocal images of the synovia from WT and IRF5KO mice show fewer neutrophils in IRF5KO mice, in line with FACS data (CD68, cyan; VSIG4, magenta; CD177, green; nuclei, gray; scale bar = 50 µm). B, bone; SC, synovial cavity. (D) Representative images of Fast Green and Safranin O–stained knee sections from a single experiment in WT and CX3CR CreER IRF5 fl/fl at day 7 p.c. (upper panels, articular surface region; lower panels, patellar region; asterisks indicate bone erosion, arrows synovial hyperplasia, and arrowheads fat pad infiltration; coronal orientation; scale bar = 300 µm). (E) Clinical scoring of respective sections for synovial hyperplasia, bone erosion, and BM proliferation shows improved pathology in lining macrophage–specific IRF5KO mice.

Article Snippet: Total macrophages were labeled with either unconjugated rat anti-CD68 (Bio-Rad) and goat anti-rat Brilliant Violet 421 secondary antibody (BD Biosciences), or anti-CD68 directly conjugated to Brilliant Violet 421 (BD Biosciences), whereas goat-derived anti-VSIG4 (Bio-Techne) followed by donkey anti-goat Alexa Fluor 647 (Life Technologies) was used to delineate lining macrophages.

Techniques: Activation Assay, Staining, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Systemic Lipopolysaccharide Challenge Induces Inflammatory Changes in Rat Dorsal Root Ganglia: An Ex Vivo Study

doi: 10.3390/ijms232113124

Figure Lengend Snippet: Nuclear translocation of inflammatory transcription factors nuclear factor interleukin 6 (NF-IL6) and signal transducer and activator of transcription 3 (STAT3) in cultured dorsal root ganglia (DRG) cells. Immunocytochemistry was performed in cultured DRG cells obtained from PBS- and LPS-treated rats to examine activation of inflammatory transcription factors. ( A ) In CD-68-positive macrophages (green) an enhanced nuclear signal of NF-IL6 (red) was detectable in rats treated with LPS ( A.2 ) compared to controls ( A.1 ). Calculating the mean nuclear intensity of the signal in the area of the nucleus (blue), a significant increase is detectable ( A.3 ). ( B ) The STAT3 signal (red) was mainly detectable in MAP-positive neurons (green) of DRG cultures of LPS-treated animals ( B.2 ) and controls ( B.1 ). Calculating the mean nuclear intensity of the signal in the area of neuronal nuclei (blue), a significant difference is detectable ( B.3 ). Bars represent the mean ± SEM. ‘ n ’ represents the number of investigated cells of the respective cell type. Scale bars present 10 µm. **: p < 0.01; ****: p < 0.0001. PBS: phosphate buffered saline, LPS: lipopolysaccharide.

Article Snippet: For immunocytochemical identification of cell types, the following monoclonal antibodies or polyclonal antisera were used: CD-68 for macrophages (mouse anti rat-CD-68; 1:1000; AbD Serotec, Oxford, UK) and microtubule-associated protein 2a+b for neurons (mouse AP-20 anti-MAP2a+b; 1:600; Sigma-Aldrich Chemie GmbH).

Techniques: Translocation Assay, Cell Culture, Immunocytochemistry, Activation Assay, Saline

Journal: PLoS ONE

Article Title: Vascularization and biocompatibility of poly(ε-caprolactone) fiber mats for rotator cuff tear repair

doi: 10.1371/journal.pone.0227563

Figure Lengend Snippet: Representative histological staining of neutrophilic granulocytes (A-C) 14 days after implantation into the dorsal skinfold chamber of BALB/c mice using naphtol-AS-D-chloroacetate-esterase (CAE) staining. Representative histological staining for detection of macrophages and monocytes (D-I) 14 days after implantation into the dorsal skinfold chamber of BALB/c mice. (D-F) Immunohistochemical detection of CD68 for detection of macrophages. (G-I) Negative controls. Areas of the implant are marked (#) .

Article Snippet: For detection of capillaries, endothelial cells were immunohistochemically stained using a rabbit anti-mouse CD31 antibody (LifeSpan Biosciences, Seattle, USA purchased from BIOZOL Diagnostica Vertrieb GmbH, Eching, Germany); a rat anti-mouse CD68 antibody (Acris Antibodies GmbH, Herford, Germany) was used to detect macrophages; a rabbit anti-mouse CSF1R antibody (OriGene Technologies, Inc., Rockville, USA) was used for detection of macrophages in general; a rat anti-mouse CD86 antibody (OriGene Technologies, Inc., Rockville, USA) was used to detect macrophages with predominantly pro-inflammatory actions; a rabbit anti-mouse CD11b antibody (OriGene Technologies, Inc., Rockville, USA) was used to detect monocytes and a rat anti-mouse CD3 antibody (OriGene Technologies, Rockville, Inc., USA) was used to detect T-cells.

Techniques: Staining, Immunohistochemical staining