Journal: Nature Metabolism

Article Title: Brown adipose tissue-derived Nrg4 alleviates endothelial inflammation and atherosclerosis in male mice

doi: 10.1038/s42255-022-00671-0

Figure Lengend Snippet: AKO and DKO mice aged 6 weeks were fed a WD for 12 weeks (6 mice in each group). a , b , The vasodilatation reaction induced by Ach ( a ) and SNP ( b ) ( n = 4). c , Representative images of en face atherosclerotic lesions. d , Quantitative analysis of c ( n = 6). e , Representative images of the cross-sectional area of the aortic root ( n = 6). Scale bars, 200 μm. f , Quantitative analysis of e . g , Representative immunohistochemical staining images of VSMCs (α-smooth muscle actin (α-SMA)), collagen (Masson), macrophages (anti-CD68) and T lymphocytes (anti-CD3) in aortic plaques. Scale bar, 20 μm. h , Quantitative analysis of g ( n = 6). I , Representative hematoxylin and eosin (H&E) staining images of plaque. Dashed lines indicate the contour of necrotic lipid core; scale bars, 50 μm. j , The quantitative analysis of necrotic core and fibrous cap thickness. The necrotic core is presented as a percentage of lesion area and the fibrous cap thickness is measured at the midpoint and shoulder regions of each lesion and quantified as the ratio of cap thickness to lesion size ( n = 6). AU, arbitrary units. k , The expressions of matrix metalloproteinase (MMP) 2 and MMP9 in mice aortic tissue. l , Quantitative analysis of k ( n = 6). m , The mRNA levels of adhesion molecules (VCAM-1, ICAM-1 and E-selectin) and inflammation (TNF-α, IL-1β and IL-6) in MAECs of mice ( n = 6). Statistical significance was calculated using two-sided Student’s t -tests. The data are presented as mean ± s.e.m. * P < 0.05; # P < 0.01.

Article Snippet: Briefly, aortic arch sections were incubated with primary antibodies for α-SMA (1:2,000 dilution, Servicebio, GB13044), Col3a1 (1:200 dilution, Abcam, ab6310), CD68 (1:100 dilution, ABclonal, A20803) and CD3 (1:100 dilution, ABclonal, A19017).

Techniques: Immunohistochemical staining, Staining

Journal: Nature Metabolism

Article Title: Brown adipose tissue-derived Nrg4 alleviates endothelial inflammation and atherosclerosis in male mice

doi: 10.1038/s42255-022-00671-0

Figure Lengend Snippet: Mice aged 6 weeks were fed a WD for 12 weeks (6 mice in each group). a , The vasodilatation reaction induced by Ach ( n = 6). b , Representative images of TUNEL staining in sections of thoracic aortas ( n = 6). Arrows indicate CD31/TUNEL colocalization. Scale bars, 100 μm. c , The percentage of apoptotic endothelial cells ( n = 6). d , Representative electron microscopy images of endothelium. Scale bars, 5 μm. e , Representative images of en face atherosclerotic lesions. f , Quantitative analysis of e ( n = 6). g , Representative images of the cross-sectional area of the aortic root ( n = 6). Scale bars, 200 μm. h , Quantitative analysis of g . i , Representative immunohistochemical staining images of VSMCs (α-SMA), collagen (Masson), macrophages (anti-CD68) and T lymphocytes (anti-CD3) in aortic plaques. Scale bar, 20 μm. j , Quantitative analysis of I ( n = 6). k , Representative H&E staining images of plaque. Dashed lines indicate the contour of the lipid core. Scale bars, 50 μm. l , The quantitative analysis of necrotic core and fibrous cap thickness. The necrotic core was presented as a percentage of lesion area and the fibrous cap thickness was measured at the midpoint and shoulder regions of each lesion and quantified as the ratio of cap thickness to lesion size ( n = 6). m , The expressions of MMP2 and MMP9 in mice aortic tissue. n , Quantitative analysis of m ( n = 6). Statistical significance was calculated using two-sided Student’s t -tests. The data are presented as the mean ± s.e.m. * P < 0.05; # P < 0.01.

Article Snippet: Briefly, aortic arch sections were incubated with primary antibodies for α-SMA (1:2,000 dilution, Servicebio, GB13044), Col3a1 (1:200 dilution, Abcam, ab6310), CD68 (1:100 dilution, ABclonal, A20803) and CD3 (1:100 dilution, ABclonal, A19017).

Techniques: TUNEL Assay, Staining, Electron Microscopy, Immunohistochemical staining

Journal: Nature Metabolism

Article Title: Brown adipose tissue-derived Nrg4 alleviates endothelial inflammation and atherosclerosis in male mice

doi: 10.1038/s42255-022-00671-0

Figure Lengend Snippet: (A) Representative images of TUNEL staining in sections of thoracic aortas. TUNEL (apoptotic cells, red), anti-CD31 (endothelial cells, green), DAPI (nuclei, blue). Arrows indicate CD31/TUNEL colocalization. Scale bars, 10μm. (B) The percentage of apoptotic endothelial cells (n = 6). (C) The aortic vasodilatation induced by Ach in mice aged 24 weeks (n = 6). (D) Representative electron microscopy images of endothelium in mice. Scale bars, 5μm (n = 6). (E) The mRNA levels of adhesion molecules (VCAM-1, ICAM-1 and E-selectin) and inflammation (TNF-α, IL-1β and IL-6) in MAECs of mice aged 24 weeks (n = 6). (F) Body weight (n = 6) after BATT weekly (n = 6). (G) The lipid profiles of mice aged 24 weeks among different groups (n = 6). (H) Representative images of en face atherosclerotic lesion areas in AKO and DKO mice. (I) Representative images of the cross-sectional area of the aortic root in AKO and DKO mice. Scale bars, 200μm. ( J ) Quantitative analysis of ( H ) (n = 6). ( K ) Quantitative analysis of (I) (n = 6). ( L ) Representative immunohistochemical staining images of VSMCs [α–smooth muscle actin (α-SMA)], collagen (Masson), macrophages (anti-CD68), and T lymphocytes (anti-CD3) in aortic plaques. Scale bar, 20 μm. (M) Quantitative analysis of (L) (n = 6). ( N ) Representative H&E staining images of plaque. A necrotic lipid core is indicated by *; dashed lines indicate the contour of the lipid core; scale bars: 50 μm. ( O ) The quantitative analysis of necrotic core and fibrous cap thickness. The necrotic core was presented as a percentage of lesion area and the fibrous cap thickness was measured at the midpoint and shoulder regions of each lesion and quantified as the ratio of cap thickness to lesion size (n = 6). ( P ) The expressions of MMP2 and MMP9 in mice aortic tissue. ( Q ) Quantitative analysis of (P) (n = 6). For b, c, e, f, and g, P values were calculated by one-way ANOVA with Tukey’s multiple-comparison test. For j, k, m, o and q, P values were calculated by two-sided t -test. Data were shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Briefly, aortic arch sections were incubated with primary antibodies for α-SMA (1:2,000 dilution, Servicebio, GB13044), Col3a1 (1:200 dilution, Abcam, ab6310), CD68 (1:100 dilution, ABclonal, A20803) and CD3 (1:100 dilution, ABclonal, A19017).

Techniques: TUNEL Assay, Staining, Electron Microscopy, Immunohistochemical staining

Journal: Nature Metabolism

Article Title: Brown adipose tissue-derived Nrg4 alleviates endothelial inflammation and atherosclerosis in male mice

doi: 10.1038/s42255-022-00671-0

Figure Lengend Snippet: AAV-Nrg4 or AAV-Zsgreen at a dose of 1 × 10 12 viral genomes were injected into the BAT in the interscapular region of KO mice at aged 6 weeks. a , Representative images of en face atherosclerotic lesion areas in AKO and DKO mice. b , Representative images of the cross-sectional area of the aortic root in AKO and DKO mice. Scale bars, 200 μm. c , Quantitative analysis of a ( n = 6). d , Quantitative analysis of b ( n = 6). e , Representative immunohistochemical staining images of VSMCs (α-SMA), collagen (Masson), macrophages (anti-CD68) and T lymphocytes (anti-CD3) in aortic plaques. Scale bar, 20 μm. f , Quantitative analysis of e ( n = 6). g , Representative H&E staining images of plaque. Dashed lines indicate the contour of the lipid core. Scale bars, 50 μm. h , The quantitative analysis of necrotic core and fibrous cap thickness. The necrotic core is presented as a percentage of lesion area and the necrotic lipid core and the fibrous cap thickness is measured at the midpoint and shoulder regions of each lesion and quantified as the ratio of cap thickness to lesion size ( n = 6). i , The expressions of MMP2 and MMP9 in mice aortic tissue. j , Quantitative analysis of i ( n = 6). Statistical significance was calculated using two-sided Student’s t -tests or one-way ANOVA (Tukey’s multiple-comparison test). Data are shown as mean ± s.e.m. * P < 0.05 versus AKO-AAV (Zsgreen); ** P < 0.01 versus AKO-AAV (Zsgreen); *** P < 0.001 versus AKO-AAV (Zsgreen); # P < 0.05 versus DKO-AAV (Zsgreen); ## P < 0.01 versus DKO-AAV (Zsgreen); ### P < 0.001 versus DKO-AAV (Zsgreen); NS, not significant.

Article Snippet: Briefly, aortic arch sections were incubated with primary antibodies for α-SMA (1:2,000 dilution, Servicebio, GB13044), Col3a1 (1:200 dilution, Abcam, ab6310), CD68 (1:100 dilution, ABclonal, A20803) and CD3 (1:100 dilution, ABclonal, A19017).

Techniques: Injection, Immunohistochemical staining, Staining

Journal: Nature Metabolism

Article Title: Brown adipose tissue-derived Nrg4 alleviates endothelial inflammation and atherosclerosis in male mice

doi: 10.1038/s42255-022-00671-0

Figure Lengend Snippet: Nrg4 overexpression of BAT in situ by AAV-Nrg4 was performed in DKO mice aged 6 weeks and leukocyte homing was analyzed in DKO-AAV (Zsgreen) or DKO-AAV (Nrg4) mice that were fed a WD for 12 weeks. a , The mRNA expression of the macrophage markers F4/80 and CD68 in aortas ( n = 6). b , The mRNA expression of the chemokines in aortas ( n = 6). c , d , The homing of GFP leukocytes to atherosclerotic plaques 48 h after intravenous injection into DKO-AAV (Zsgreen) and DKO-AAV (Nrg4) mice that were fed a WD for 12 weeks. c , Fluorescence micrograph of aortic root plaques. The dashed line indicates the plaque border. Inset, magnification of GFP leukocytes. DAPI (left); GFP (middle); merge (right). Scale bars, 200 μm. d , Quantification of GFP leukocytes per square millimeter of plaque ( n = 6). e , The representative images of aortic arch sections stained with CD31 (red, as an endothelial marker), VCAM-1 or ICAM-1 (green) and DAPI (blue) ( n = 6). Arrowheads indicate CD31/VCAM-1 or CD31/ICAM-1 colocalization. Scale bars, 150 μm. f , The mRNA expressions from MAECs of the aorta for VCAM-1 and ICAM-1 ( n = 6). Statistical significance was calculated using two-sided Student’s t -tests. Data are shown as mean ± s.e.m. * P < 0.001.

Article Snippet: Briefly, aortic arch sections were incubated with primary antibodies for α-SMA (1:2,000 dilution, Servicebio, GB13044), Col3a1 (1:200 dilution, Abcam, ab6310), CD68 (1:100 dilution, ABclonal, A20803) and CD3 (1:100 dilution, ABclonal, A19017).

Techniques: Over Expression, In Situ, Expressing, Injection, Fluorescence, Staining, Marker

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: Glia

Article Title: Activated microglia do not increase 18 kDa translocator protein ( TSPO ) expression in the multiple sclerosis brain

doi: 10.1002/glia.24052

Figure Lengend Snippet: Antibodies for immunohistochemistry

Article Snippet: CD68 , Rabbit , Atlas Antibody , HPA048982 , 1:4000.

Techniques:

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: 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