mouse lyve-1 antibody  (Bio-Techne corporation)

Journal: bioRxiv

Article Title: Lymphatic activation of ACKR3 signaling regulates lymphatic response after ischemic heart injury

doi: 10.1101/2024.12.04.626683

Figure Lengend Snippet: A) 3-D volumetric view of the cardiac lymphatic network visualized by light-sheet imaging of Prox1 GFP mouse hearts immunostained with anti-LYVE1 and anti-GFP antibodies using the iDisco tissue clearing and staining protocol. Scalebar, 1000 µm. B-D ) Expanded cross-section views of the cardiac lymphatic vessels of Prox1 GFP mouse hearts. Low-magnification images ( B , scalebar, 1000 µm), and insets from the left ventricular free wall ( C ) and the cardiac septum (D) (bars, 300 µm) are shown. Yellow arrowheads point at cardiac lymphatic vessels penetrating into the deeper myocardial layers. Red arrowheads point at lymphatic vessels in the cardiac septum facing the right ventricle. White arrows point at the cardiac septal wall facing the left ventricle. Representative images from 3 animals are shown.

Article Snippet: Dissected organs were fixed for 24h in 4% PFA at 4°C, followed by routine whole mount immunostaining with goat anti-LYVE1 (1:200, R&D Systems, AF2125-SP) primary, and Alexa Fluor 488-conjugated donkey anti-goat (1:200, Jackson ImmunoResearch, 705-545-003) secondary antibodies.

Techniques: Imaging, Staining

Journal: bioRxiv

Article Title: Lymphatic activation of ACKR3 signaling regulates lymphatic response after ischemic heart injury

doi: 10.1101/2024.12.04.626683

Figure Lengend Snippet: A,B) LYVE-1-positive cardiac lymphatic vessels and ACKR3 activation detected by H2B-GFP expression in ACKR3-TangoGFP mice 2 days post sham surgery (A) or LAD ligation (B) . Expanded cross-section views are shown. Yellow arrowheads point at H2B-GFP signal overlapping with LYVE1 immunostaining in epicardial LYVE1-positive lymphatic structures in the left basal ventrolateral region of the heart of LAD-ligated mice, adjacent to the ligation site. Bars, 1000 µm. C) Rendered pseudocolored images showing LYVE1-positive cardiac lymphatic vessels (green), ACKR3 activation in LYVE1-negative (blue) and LYVE1-positive (magenta) cells in the basal and apical ventrolateral segments of the left ventricle free wall of ACKR3-TangoGFP mice 2 days post sham surgery or LAD ligation. Bars, 200 µm. Representative images from 4-5 animals per group are shown. D) Relative lymphatic GFP signal (calculated as [N(GFP) LYVE1+ / N(GFP) LYVE1- ] injury site / [N(GFP) LYVE1+ /N(GFP) LYVE1+ ] uninjured tissue ) in the basal and apical ventrolateral segments of the left ventricle free wall compared to the uninjured apical septal wall facing the right ventricle in ACKR3-TangoGFP mice 2-days after LAD ligation or sham surgery and the average of the values from these two areas. Each symbol represents quantification from one mouse. Mice in the LAD ligated group are identified by numbering. Unpaired t-test, N=4-5 per group.

Article Snippet: Dissected organs were fixed for 24h in 4% PFA at 4°C, followed by routine whole mount immunostaining with goat anti-LYVE1 (1:200, R&D Systems, AF2125-SP) primary, and Alexa Fluor 488-conjugated donkey anti-goat (1:200, Jackson ImmunoResearch, 705-545-003) secondary antibodies.

Techniques: Activation Assay, Expressing, Ligation, Immunostaining

Journal: bioRxiv

Article Title: Lymphatic activation of ACKR3 signaling regulates lymphatic response after ischemic heart injury

doi: 10.1101/2024.12.04.626683

Figure Lengend Snippet: A,B) LYVE-1-positive cardiac lymphatics and ACKR3 activation detected by H2B-GFP expression in ACKR3-Tango-GFP ; Ramp3 -/- mice 2-days post sham surgery (A) or LAD ligation (B) . Representative expanded cross-section views from 2 animals per group are shown. Bars, 1000 µm. C) 3-D high magnification views showing LYVE1-positive lymphatics and ACKR3 activation detected by H2B-GFP signal in the basal and apical ventrolateral segments of the left ventricle free wall of ACKR3-Tango-GFP ; Ramp3 -/- mice 2-days post sham surgery or LAD ligation. Bars, 200 µm. D, E) Representative Western Blot image showing phosphorylation of ERK, AKT, and CREB (D) and quantification of p-ERK/t-ERK ratios (E) in hLECs treated with adrenomedullin for 5, 10, 20, 30, 60 minutes after treatment with control human beta globin (shHGB) or shRAMP3 constructs. N = 4 per group. Two way ANOVA; Šidak’s multiple comparisons test compared to shHGB.

Article Snippet: Dissected organs were fixed for 24h in 4% PFA at 4°C, followed by routine whole mount immunostaining with goat anti-LYVE1 (1:200, R&D Systems, AF2125-SP) primary, and Alexa Fluor 488-conjugated donkey anti-goat (1:200, Jackson ImmunoResearch, 705-545-003) secondary antibodies.

Techniques: Activation Assay, Expressing, Ligation, Western Blot, Control, Construct

Journal: bioRxiv

Article Title: Lymphatic activation of ACKR3 signaling regulates lymphatic response after ischemic heart injury

doi: 10.1101/2024.12.04.626683

Figure Lengend Snippet: A) Venn diagram summarizing the transcriptomic differences in cardiac LECs isolated from male Ackr3 fl/fl and Ackr3 ΔLyve mice one week post LAD ligation. B) Differentially expressed genes in cardiac LECs upon LAD ligation that are specific to ACKR3-deficient mice. Positive fold change values represent upregulation in LAD ligated Ackr3 ΔLyve LECs. C) Comparison of gene expression levels of identified ACKR3-specific LAD-triggered genes between LAD ligated Ackr3 ΔLyve and Ackr3 fl/fl mice. Positive fold change values represent upregulation in LAD ligated Ackr3 ΔLyve LECs. D) Visualization of LYVE1-positive cardiac lymphatic vessels of male Ackr3 fl/fl and Ackr3 ΔLyve whole mount hearts 6- and 28-days post LAD ligation. Representative images of from 3-4 animals per group. Bars, 500 µm. E,F) Quantification of lymphatic vessel length (E) and branching index (number of branches divided by lymphatic vessel length) (F) per field of view. N=3-4 per group. Two-way ANOVA, Dunnett’s post-hoc test. G) LYVE1 (cyan) and CD45 (magenta) staining of the infarct zone and peri-infarct zones of male Ackr3 fl/fl and Ackr3 ΔLyve hearts 28-days post LAD ligation. Yellow arrows point at LYVE1-positive lymphatic structures in the injured tissues of the infarct and peri-infarct zone of hearts. White arrowheads point at epicardial lymphatic structures. Representative images from N=3-6 mice per group. Bars, 200 µm. H,I) Area-adjusted lymphatic vessel counts in the infarct zone (H) and percent area of the infarct zone occupied by lymphatic vessels (I) in male Ackr3 fl/fl and Ackr3 ΔLyve hearts 28-days post LAD ligation. N= 3-6 per group. Unpaired Welch’s unequal variances t-test.

Article Snippet: Dissected organs were fixed for 24h in 4% PFA at 4°C, followed by routine whole mount immunostaining with goat anti-LYVE1 (1:200, R&D Systems, AF2125-SP) primary, and Alexa Fluor 488-conjugated donkey anti-goat (1:200, Jackson ImmunoResearch, 705-545-003) secondary antibodies.

Techniques: Isolation, Ligation, Comparison, Expressing, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: EphrinB2-mediated CDK5/ISL1 pathway enhances cardiac lymphangiogenesis and alleviates ischemic injury by resolving post-MI inflammation

doi: 10.1038/s41392-024-02019-4

Figure Lengend Snippet: Overexpression of EphrinB2 promotes cardiac lymphangiogenesis and reduces cardiac inflammation post-MI. a Whole mount imaging showing endogenous tdTomato (white) fluorescence in the hearts from Lyve1 -Cre; Rosa26 -tdTomato mice after sham or MI operation. Magnified views of yellow dashed boxes are shown in the right lane. The yellow arrows highlight the lymphatics in the infarct area and border zone. Scar bar: 1 mm. b (Left) Representative immunofluorescence staining images showing myocardium co-stained by CD31 (red), VEGFR3 (green), and DAPI (blue) of mice injected with AAV- Efnb2 or AAV-NC after MI operation. Scar bar: 50 μm. (Right) Quantification of VEGFR3 + lymphatics (n = 5 per group). c (Left) Representative immunoblotting images showing the protein levels of VEGFR3 and LYVE1 in the hearts from indicated groups. (Right) Quantification of immunoblotting results normalized to Actin and presented relative to the AAV-NC group (n = 5 per group). d Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis determining the mRNA expressions of pro-inflammatory genes in the hearts of mice that were injected with AAV-NC or AAV- Efnb2 at day 7 post-operation (n = 5 per group). e (Left) Fluorescence-activated cell sorter (FACS) analysis of CD45 + CD11b + Ly6G − F4/80 + Ly6C high macrophages in the murine hearts at day 7 post-MI. (Right) Quantification of CD45 + CD11b + Ly6G − F4/80 + Ly6C high macrophages as the percentage of CD45 + cells (n = 5 per group). f Representative immunofluorescence staining images showing the myocardium co-stained by CD68 (red), VEGFR3 (green), and DAPI (blue) in indicated groups. Scar bar: 20 μm. g Schematic diagram depicting the experimental strategy for Evans blue injection from cardiac apex to lymph nodes through the lymphatic system. h Representative heart images of Evans blue in the mediastinal lymph nodes and lymphatics. Scar bar: 1 mm. i (Left) Representative immunofluorescence staining images showing mediastinal lymph node co-stained by CD68 (red) and DAPI (blue) in indicated groups. Scar bar: 50 μm. (Right) Quantification of CD68 + macrophages as the percentage of cells in MLNs. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. c , d by one-way ANOVA with Tukey post-hoc test, b by unpaired Student’s test, and e and i by Mann–Whitney U test. MLN mediastinal lymphatic node, and LV lymphatic vessel

Article Snippet: The antibodies utilized in this study were as follows: EphrinB2 (1 μg/ml; AF496, R&D system), VEGFR3/FLT4 (0.1 μg/ml; AF743, R&D system), LYVE1 (0.25 μg/ml; AF2125, R&D system), ISL1 (1:1000; ab86501, Abcam), CDK5 (1:2000; ab40773, Abcam), Pan phosphor-Serine/Threonine (1:1000; AP1745, ABclonal), β-actin (1:5000; KC-5A08, KangChen).

Techniques: Over Expression, Imaging, Fluorescence, Immunofluorescence, Staining, Injection, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, MANN-WHITNEY

Journal: Signal Transduction and Targeted Therapy

Article Title: EphrinB2-mediated CDK5/ISL1 pathway enhances cardiac lymphangiogenesis and alleviates ischemic injury by resolving post-MI inflammation

doi: 10.1038/s41392-024-02019-4

Figure Lengend Snippet: Lyve1 deficiency abrogates the anti-inflammatory effects of EphrinB2 post-MI. a Schematic diagram depicting the experimental strategy for EphrinB2 overexpression in Lyve1 −/− mice and their WT littermates. b Representative histological images from Lyve1 −/− mice and their WT littermates that were injected with AAV-NC or AAV- Efnb2 assessed by Masson Trichrome staining. Magnified views of black boxes are shown in the bottom lane. Scar bar: 1 mm. c Representative M-mode echocardiographic images showing the cardiac function of Lyve1 −/− mice and their WT littermates that were injected with AAV-NC or AAV- Efnb2 . The yellow lines indicate the endocardium of the anterior and posterior walls at mid-papillary muscle level. d – f Quantification of echocardiographic parameters in c (LVEF, LVIDs, and LVIDd, n = 6 per group). g Quantification of infarct size of myocardium in b (n = 5 per group). h Quantification of apoptotic cells as percentage of all cells in i . i Representative TUNEL staining images showing cell apoptosis in indicated groups. Scar bar: 50 μm. j Representative immunofluorescence staining images showing the myocardium co-stained by CD31 (red), VEGFR3 (green), and DAPI (blue) in indicated groups. Scar bar: 50 μm. k Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis determining the mRNA expressions of pro-inflammatory genes in the hearts in indicated groups at day 7 post-MI (n = 5 per group). l (Left) Fluorescence-activated cell sorter (FACS) analysis of CD45 + CD11b + Ly6G − F4/80 + Ly6C high macrophages in the murine hearts at day 7 post-MI. m Quantification of the density of VEGFR3 + lymphatics in j . n Quantification of CD45 + CD11b + Ly6G − F4/80 + Ly6C high macrophages as the percentage of CD45 + cells in l (n = 4 per group). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. d–h , k , and m by one-way ANOVA with Tukey post-hoc test, and n by Mann-Whitney U test. LVEF left ventricular ejection fraction, LVIDs left ventricular internal dimension during systole, LVIDd left ventricular internal dimension during diastole, DAPI 4’,6-diamidino-2-phenylindole, HE hematoxylin and eosin, and TUNEL terminal deoxynucleotidyltransferase-mediated dUTPbiotin nick end labeling

Article Snippet: The antibodies utilized in this study were as follows: EphrinB2 (1 μg/ml; AF496, R&D system), VEGFR3/FLT4 (0.1 μg/ml; AF743, R&D system), LYVE1 (0.25 μg/ml; AF2125, R&D system), ISL1 (1:1000; ab86501, Abcam), CDK5 (1:2000; ab40773, Abcam), Pan phosphor-Serine/Threonine (1:1000; AP1745, ABclonal), β-actin (1:5000; KC-5A08, KangChen).

Techniques: Over Expression, Injection, Staining, TUNEL Assay, Immunofluorescence, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Fluorescence, MANN-WHITNEY, End Labeling