goat anti lyve1  (R&D Systems)

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