Journal: Journal of Virology

Article Title: Brain Large Artery Lymphocytic Inflammation and Human Immunodeficiency Virus-Related Brain Arterial Remodeling

doi: 10.1128/JVI.00081-18

Figure Lengend Snippet: Intima CD3 + /CD68 + cell phenotypes and markers of brain arterial remodeling a

Article Snippet: Briefly, anti-CD68 + antibody (primary, 1:100, catalog number M0814, from Dako Corp., Carpinteria, CA; secondary, biotinylated horse anti-mouse IgG antibody, catalog number BA-2000, from Vector Laboratories, Burlingame, CA) binding to arteries was counterstained with hematoxylin and visualized using the diaminobenzidine kit ( 10 ).

Techniques: Isolation

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: American Journal of Respiratory Cell and Molecular Biology

Article Title: Mesenchymal Stem Cell Extracellular Vesicles Reverse Sugen/Hypoxia Pulmonary Hypertension in Rats

doi: 10.1165/rcmb.2019-0154OC

Figure Lengend Snippet: (A) Immunohistochemical staining of Cd206+ macrophages in lungs of rats treated with normoxia, (B and C) SuHx plus vehicle (B), and SuHx plus MSC-EV (C). Scale bars: 50 μm. (D–F) Average number of Cd206+ per 10× field (D), Cd68+ total rat macrophages per 10× field (E), and ratio of Cd206+/Cd68+ macrophages per 10× lens objective field of view (F) in lung sections from rats treated with Nx, SuHx plus vehicle, or SuHxEV. *P < 0.05, **P < 0.01, and ****P < 0.0001.

Article Snippet: Lungs were kept in 4% paraformaldehyde until embedded in paraffin; sectioned in 5-μm slices; and stained with antibody against rat ACTA2 (ab5694; Abcam) to assess pulmonary vascular remodeling, antibody against rat von Willebrand factor (vWF) (ab6994; Abcam) to assess distal vessel count, and antibodies against rat Cd206 (ab64693; Abcam) or Cd68 (ED1; Bio-Rad Laboratories) for assessment of macrophage recruitment.

Techniques: Immunohistochemical staining, Staining

Journal: Circulation Research

Article Title: miR33 Inhibition Overcomes Deleterious Effects of Diabetes Mellitus on Atherosclerosis Plaque Regression in Mice

doi: 10.1161/circresaha.115.304164

Figure Lengend Snippet: Figure 2. α-miR33 treatment restores regression in diabetic mice. Aortic roots from baseline and the regression groups were sectioned, fixed, and stained for CD68 (A) and collagen (B). Representative pictures of CD68 immunostaining (A, magnification ×20) and picrosirius red staining (B, under white and polarized light) of collagen (magnification ×10) are shown for each group. The areas of the plaques occupied by CD68+ cells and collagen (the latter as detected by polarized light) were quantified by Image Pro Plus Software and displayed in the graphs. Results are expressed as the percentage of plaque area. ^P≤0.05 vs baseline, #P≤0.05, ###P≤0.001 vs con α-miR normoglycemic; ***P≤0.001 vs α-miR33 diabetic.

Article Snippet: For immunostaining of CD68 (macrophage marker), slides were fixed in 100% acetone and exposed to primary anti-CD68 antibody (Serotec), followed by biotinylated secondary antibody (Vector Laboratories), with visualization using a Vectastain ABC kit (Vector Laboratories).

Techniques: Staining, Immunostaining, Software

Journal: Frontiers in Nutrition

Article Title: Tissue specific role of ABCA1 in lung cholesterol homeostasis under high-cholesterol diet

doi: 10.3389/fnut.2025.1649407

Figure Lengend Snippet: Subcellular localization of ABCA1 in lung tissue. (A) Co-staining of ABCA1 (red) and SFTPC (green, alveolar type II marker); nuclei stained with DAPI (blue). (B) Co-staining of ABCA1 (red) and CD68 (green, macrophage marker) to determine cell-type-specific expression. n = 3–5 mice/group.

Article Snippet: Finally, DAPI was applied in the dark for 5 min, and the sections were sealed with an anti-fade mounting medium CD68 (1:100, boster), SFTPC (1:100, Proteintech), ABCA1(1:100, Proteintech).

Techniques: Staining, Marker, Expressing

Journal: Scientific Reports

Article Title: Dietary wheat amylase trypsin inhibitors promote features of murine non-alcoholic fatty liver disease

doi: 10.1038/s41598-019-53323-x

Figure Lengend Snippet: ATI feeding increases liver macrophage numbers and their M1- vs M2-type polarization. ( A – C ) Immunohistochemistry and quantitative morphometry for CD68 and YM-1 positive cells (original magnification 40x). ( D ) Ratio of total (CD68+) vs M2-type (Ym-1+) macrophages. ( E ) CD11b+ F4/80+ macrophage subset (% of CD45 positive total immune cells) as determined by FACS analysis. Comparisons by ANOVA; data are means ± SEM for 10 representative sections per mouse and 7–10 mice per group; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Tissue was blocked with 5% normal donkey serum, and subsequently incubated with primary antibodies of to CD68 (1:100, Biozol, clone: FA-11), CD86 (1:100, Abcam, cat no: ab119857), and MHC-II (1:100, Abcam, Cat no: 180779) for 2 hrs at room temperature and finally incubated with respective Alexa-flour 488 labelled secondary antibodies.

Techniques: Immunohistochemistry

Journal: Scientific Reports

Article Title: Dietary wheat amylase trypsin inhibitors promote features of murine non-alcoholic fatty liver disease

doi: 10.1038/s41598-019-53323-x

Figure Lengend Snippet: ATI feeding increases hepatic pro-inflammatory and macrophage M1- vs M2-type gene expression. ( A – F ) Hepatic transcript levels of cd68, tnfa, il1b, il6, arg1 and ym1. Comparisons by ANOVA; data are expressed as means ± SEM for 7–10 mice per group; *p < 0.05, **p < 0.01.

Article Snippet: Tissue was blocked with 5% normal donkey serum, and subsequently incubated with primary antibodies of to CD68 (1:100, Biozol, clone: FA-11), CD86 (1:100, Abcam, cat no: ab119857), and MHC-II (1:100, Abcam, Cat no: 180779) for 2 hrs at room temperature and finally incubated with respective Alexa-flour 488 labelled secondary antibodies.

Techniques: Gene Expression

Journal: Scientific Reports

Article Title: Dietary wheat amylase trypsin inhibitors promote features of murine non-alcoholic fatty liver disease

doi: 10.1038/s41598-019-53323-x

Figure Lengend Snippet: Nutritional ATI promote central adipose tissue inflammation. ( A ) Crown like structures (CLS = accumulation of macrophages) in CD68+ stained sections of epididymal adipose tissue in the 4 experimental groups (original magnification 40x), the number of CD68+ CLS as determined by morphometry, and epididymal fat as % of body weight. ( B ) fat weights, and ( C ) transcript levels of cd68, il6 and il1b. Comparisons by ANOVA; data are means ± SEM for 10 representative sections per mouse and 7–10 mice per group; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Tissue was blocked with 5% normal donkey serum, and subsequently incubated with primary antibodies of to CD68 (1:100, Biozol, clone: FA-11), CD86 (1:100, Abcam, cat no: ab119857), and MHC-II (1:100, Abcam, Cat no: 180779) for 2 hrs at room temperature and finally incubated with respective Alexa-flour 488 labelled secondary antibodies.

Techniques: Staining

Journal: Scientific Reports

Article Title: Dietary wheat amylase trypsin inhibitors promote features of murine non-alcoholic fatty liver disease

doi: 10.1038/s41598-019-53323-x

Figure Lengend Snippet: ATI feeding increases intestinal macrophage and dendritic cell activation and maturation. ( A–C ) CD68, CD86 and MCH-II expressing cells in the terminal ileum; scale bar: 100 and 50 µm. ( D ) Morphometric quantification of CD68, CD86 and MHC-II positive cells. ( E ) Transcript levels of il1b, tnfα and il6. Comparisons by ANOVA; data are expressed as means ± SEM of 6 mice per group and 5 representative sections per mouse; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Tissue was blocked with 5% normal donkey serum, and subsequently incubated with primary antibodies of to CD68 (1:100, Biozol, clone: FA-11), CD86 (1:100, Abcam, cat no: ab119857), and MHC-II (1:100, Abcam, Cat no: 180779) for 2 hrs at room temperature and finally incubated with respective Alexa-flour 488 labelled secondary antibodies.

Techniques: Activation Assay, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Immune homeostasis and regulation of the interferon pathway require myeloid-derived Regnase-3

doi: 10.1084/jem.20181762

Figure Lengend Snippet: Regnase-3 –deficient mice develop hypertrophic lymph nodes. (A) Frequency of mice showing lymphadenopathy in a cohort of 24 Regnase-3 −/− mice and 24 Regnase-3 +/+ littermate controls at 3–6.5 mo of age. (B) Photography of skin-draining lymph nodes of four Regnase-3 −/− mice and their Regnase-3 +/+ littermate controls at 5 mo of age. (C) Representative photography of inguinal lymph nodes of a Regnase-3 −/− mouse at 5 mo of age. Arrow indicates hypertrophic lymph node. (D) H&E staining and immunohistochemical analysis of B cells (B220), T cells (CD3), and macrophages (F4/80) in skin-draining lymph nodes of Regnase-3 −/− mice with lymphadenopathy and Regnase-3 +/+ littermate controls (representative images from n = 3/3). Magnification of images is indicated in brackets. Bars, 1,000 µm. (E) Immunohistochemical analysis of macrophages (CD68) in skin-draining lymph nodes of Regnase-3 −/− mice with lymphadenopathy and Regnase-3 +/+ littermate controls (representative images from n = 6/6). Images of enlarged and small lymph nodes are taken from the identical Regnase-3 −/− mouse. Top right: Frequency of strong positive (pos.) pixels in CD68 immunohistochemical sections of the lymph nodes was determined by Definiens software ( n = 6/6). Bars, 500 µm. (F) Top: Frequencies of B cells (CD19 + ) and T cells (CD90 + ) in enlarged and normal-sized lymph nodes of the same Regnase-3 −/− mouse and its Regnase-3 +/+ littermate control at 6 mo of age, assessed by flow cytometry (representative blots of n = 6/6). Number of total cells in lymph nodes of Regnase-3 +/+ mice and Regnase-3 −/− littermates ( n = 6/6). Bottom: Frequencies of B cells (CD19 + ), T cells (CD90 + ), CD4 + and CD8 + T cells, and CD11b + cells in enlarged lymph nodes of Regnase-3 −/− mice and their Regnase-3 +/+ littermate controls at 6 mo of age, assessed by flow cytometry ( n = 6/6). Data are represented as mean ± SEM and were compared by Mann–Whitney U test (*, P ≤ 0.05; **, P ≤ 0.01; ns, not significant).

Article Snippet: Staining antibodies were anti-B220 (RA3-6B2, rat-IgG2a; BD), anti-CD3 (SP7, rabbit IgG; Zytomed), anti-F4/80 (BM8, rat-IgG2a; Linaris), anti-MHC-II (M5/114.15.2, rat IgG; Novus Biologicals), anti-CD68 (Ab125212, rabbit IgG; Abcam) anti-ki67 (SP6, rabbit IgG; Thermo Fisher Scientific), and anti-Tyr701-phospho-STAT1 (58D6, rabbit IgG; Cell Signaling).

Techniques: Staining, Immunohistochemical staining, Software, Flow Cytometry, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: Immune homeostasis and regulation of the interferon pathway require myeloid-derived Regnase-3

doi: 10.1084/jem.20181762

Figure Lengend Snippet: Systemic IFN signaling in Regnase-3 −/− mice. (A) CD19 + B cells were isolated from enlarged lymph nodes of Regnase-3 −/− mice and their Regnase-3 +/+ littermates ( n = 3/3), and RNA was isolated and subjected to RNA sequencing. Heatmap of RNA sequencing data for all significantly up-regulated (≥2 log2 fold) genes in Regnase-3 −/− B cells is shown. GO term association to “response to IFNβ” and “response to IFNγ” is indicated for each gene. (B) Serum cytokines in Regnase-3 −/− mice and Regnase-3 +/+ littermates at 6 mo of age ( n = 8/8), assessed by multiplex assay. (C) Stat1 mRNA expression in tissues from Regnase-3 +/+ and Regnase-3 −/− mice at 8 mo of age, assessed by quantitative RT-PCR, normalized to Hprt relative (rel.) to their expression in Regnase-3 +/+ mice ( n = 5/5). (D) Left: Immunohistochemical analysis of MHC-II in liver sections of Regnase-3 −/− mice with lymphadenopathy and Regnase-3 +/+ controls (representative images). Magnification of images is indicated in brackets. Bars, 250 µm. Right: Frequency of strong positive pixels in MHC-II immunohistochemical sections of lung, kidney, and liver were determined by Definiens software ( n = 6 Regnase-3 −/− mice and 6 Regnase-3 +/+ littermate controls). (E) Frequency of MHC-II–positive macrophages (CD68 + ) of all CD68 + macrophages in the lung of Regnase-3 −/− mice with lymphadenopathy and their Regnase-3 +/+ littermate controls, assessed in immunohistochemical, consecutive sections ( n = 5/6). (F) Left: Immunohistochemical analysis of pSTAT1 in skin-draining lymph nodes from Regnase-3 −/− mice with lymphadenopathy and Regnase-3 +/+ littermate controls (representative images). Right: Frequency of strong positive pixels in pSTAT1 immunohistochemical sections of lymph nodes, determined by Definiens software ( n = 6 Regnase-3 −/− mice and 6 Regnase-3 +/+ littermate controls). Bars, 500 µm. Data are represented as mean ± SEM and were compared by Mann–Whitney U test (*, P ≤ 0.05; **, P ≤ 0.01; ns, not significant).

Article Snippet: Staining antibodies were anti-B220 (RA3-6B2, rat-IgG2a; BD), anti-CD3 (SP7, rabbit IgG; Zytomed), anti-F4/80 (BM8, rat-IgG2a; Linaris), anti-MHC-II (M5/114.15.2, rat IgG; Novus Biologicals), anti-CD68 (Ab125212, rabbit IgG; Abcam) anti-ki67 (SP6, rabbit IgG; Thermo Fisher Scientific), and anti-Tyr701-phospho-STAT1 (58D6, rabbit IgG; Cell Signaling).

Techniques: Isolation, RNA Sequencing Assay, Multiplex Assay, Expressing, Quantitative RT-PCR, Immunohistochemical staining, Software, MANN-WHITNEY