Journal: The EMBO Journal

Article Title: The chemokine receptors ACKR2 and CCR2 reciprocally regulate lymphatic vessel density

doi: 10.15252/embj.201488887

Figure Lengend Snippet: (i) Whole-mount anti-podoplanin (red) immunostaining of ear lymphatic vessels using WT (left-hand panel), and ACKR2-KO (right-hand panel), CD11cYFP mice for simultaneous detection of CD11c + myelomonocytic cells (green). Shown are 3D transparent projection images generated from a thickness (Z-stacks) of 13 μm (left-hand panel) and 15 μm (right-hand panel). Scale bars, 100 μm. (ii) Quantification of numbers of CD11c + cells in WT and ACKR2-KO cartilage-free ear sheets. Each point represents the mean of cell counts from at least 3 FOVs from each mouse ear imaged using a Zeiss EC Plan-Neofluar 5× /0.16 M27 lens. Data were analysed using Student's t -test. Flow cytometric quantitation of the numbers of macrophages (CD11b + F4/80 + ) in resting (acetone treated; Ace), or phorbol ester inflamed (72TPA), WT and ACKR2-KO mouse ears (7–8 mice/group with each data point representing measurements from a single mouse). Data were analysed using one-way ANOVA with Newman–Keul multiple comparison test as a post-test for differences between groups. (i) Immunostaining for macrophage proximity (CD11b, turquoise) to lymphatic vessels (podoplanin, red) in frozen ear skin sections of TPA-inflamed WT and ACKR2-KO mice. Blue represents DAPI staining of cellular nuclei. Z-stack images (at 0.6- to 1-μm intervals) for WT and ACKR2-KO mice shown here (across a thickness of up to 10 μm) were taken using a Zeiss EC Plan-Neofluar 40 × /0.75 Ph2 M27. (ii) Measured distances between macrophages and lymphatic vessel surfaces in individual z-stacks from resting (Ace) and phorbol ester inflamed (72TPA) WT and ACKR2-KO mouse ear skin frozen sections (10 mice/group with each point representing measurements from a single mouse). Data were analysed using one-way ANOVA with Newman–Keul multiple comparison test as a post-test. Immunostaining of resting 3-week-old WT mouse lymphatic vessels with antibodies to CCL2 (green) and podoplanin (red). (i) CCL2 staining; (ii) merged CCL2 and podoplanin staining; (iii) CCL2 staining with depth coding rainbow scale bar indicating the Z-axial dimensions. Confocal 3D transparent images were acquired, across a thickness of 18 μm, using a Zeiss Plan-Apochromat 63× /1.4oil Ph3 on a Zeiss LSM 510 confocal microscope. Scale bar, 20 μm. (i) High magnification imaging of adult (7 weeks old) cutaneous lymphatic vessels using antibodies to LHS image: VEGFR3 (red) and Prox-1 (blue) and RHS image: VEGFR3 (red); Prox-1 (blue) and podoplanin (green). Scale bars, 50 μm. Images were obtained using a Zeiss EC Plan-Neofluar 20× /0.50 Ph2 M27 lens. (ii) Staining of VEGF-D expression by macrophages in WT mouse ear frozen sections using anti-CD11b antibodies (cyan); anti-F4/80 antibodies (green); DAPI (blue); anti-VEGF-D antibodies (red). (iia) VEGFD-TyramideCy3; (iib) F4/80-AF488; (iic) Merged image of F4/80 and VEGFD; (iid) An overlay of all three channels. All images are maximum projection images across a 3-μm thickness obtained under an EC Plan-Neofluar 40× /0.75 Ph2 M27 lens. Scale bars, 20 μm.

Article Snippet: Forelimb skin or dorsal skin was isolated using fine forceps and scissors (Fine Science Tools, Germany) under a dissection microscope and incubated with 2 μg/ml anti-VEGFR3/anti-Lyve-1 or 1:2,000 anti-Prox-1 antibodies in cRPMI/0.05% Triton X-100 (Sigma-Aldrich, UK) overnight at 4°C with gentle agitation.

Techniques: Immunostaining, Generated, Quantitation Assay, Staining, Microscopy, Imaging, Expressing

Journal: The EMBO Journal

Article Title: The chemokine receptors ACKR2 and CCR2 reciprocally regulate lymphatic vessel density

doi: 10.15252/embj.201488887

Figure Lengend Snippet: A (i) Whole-mount staining of control and inflamed (TPA-treated) lymphatic vessel networks in adult (7–8 weeks old) mouse ears. Podoplanin staining is in green and VEGFR3 staining in red. VEGFR3 has been used here as an additional stain due to the reduction in podoplanin content in TPA-treated skin. Depth coding rainbow scale bars are included to represent the Z-axial dimensions. Scale bars, 200 μm. (ii) Higher magnification confocal imaging (63× magnification) of an intact (upper image) and a ruptured (lower image) lymphatic vessel stained using antibodies to podoplanin (red), Lyve-1 (green) and collagen IV (blue). Images were obtained using a Zeiss LSM510 using a Plan-Apochromat 63× /1.4oil Ph3 lens. Merged images shown here for these three colours are 3D maximum projection images constructed on the Imaris Bitplane software (Version 7.6.1). (iii) Ki67 staining (cyan and indicated by arrows) of resting (control: top) and inflamed (48 h post-TPA; bottom) skin of ACKR2-deficient mice. These images were obtained using an EC Plan-Neofluar 20× /0.50 Ph2 M27 lens on a Zeiss Axioimager M2 across a thickness (z-stacks) of 12 μm (control: top) or 11 μm (48 h TPA: bottom). Scale bars, 50 μm (top) and 20 μm (bottom). The images are merged and also show Lyve-1 (green) and podoplanin (red) staining. B Quantification of the numbers of ruptured vessels as assessed by counting blunt/point-ended vessel structures in resting (Ctrl) and 24 h inflamed (24) WT and ACKR2-KO mouse ears. Each data point represents the mean of 3 FOV measurements per mouse ear. C Quantification of (i) lymphatic vessel branch numbers and (ii) average distance between individual lymphatic vessels in WT and ACKR2-KO mouse ears at rest (Ctrl) and at 24 and 72 h post-TPA treatment. Each point on the graphs represents the mean of measurements from 3 FOVs per mouse ear imaged under an objective ZEISS EC Plan-Neofluar 5× /0.16 M27 on the Zeiss AxioImager M2 for quantification. D, E qPCR analysis of expression of Prox-1 (D) and VEGF-D (E) in resting (Ace) and TPA-inflamed (72) WT and ACKR2-deficient (KO) adult (7–8 weeks old) mice. Each data point represents one ear per mouse and data points from two independent experiments were pooled together. Student's t -test (E) and Mann–Whitney U -test (D) was used for the statistical analysis between groups.

Article Snippet: Forelimb skin or dorsal skin was isolated using fine forceps and scissors (Fine Science Tools, Germany) under a dissection microscope and incubated with 2 μg/ml anti-VEGFR3/anti-Lyve-1 or 1:2,000 anti-Prox-1 antibodies in cRPMI/0.05% Triton X-100 (Sigma-Aldrich, UK) overnight at 4°C with gentle agitation.

Techniques: Staining, Imaging, Construct, Software, Expressing, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: The chemokine receptors ACKR2 and CCR2 reciprocally regulate lymphatic vessel density

doi: 10.15252/embj.201488887

Figure Lengend Snippet: (i) Representative wide-field fluorescence images acquired at 10× magnification of PFA-fixed whole-mounts of E15.5 dorsal skin sheets of WT, ACKR2-KO and CCR2-KO embryos. Fixed dorsal skin sheets were stained for Prox-1 (purple) and Lyve-1 (green). Scale bars, 100 μm. White arrows indicate the localisation of macrophages along vessel walls in the ACKR2-deficient image. (ii) Axial dimensions for Prox-1-stained images. Scale bars, 100 μm. All maximum projection images were acquired using an EC Plan-Neofluar 10× /0.30 Ph1 lens on the Zeiss AxioImager M2, and all the 3D transparent projection images with depth coding rainbow scale bars were generated using Zeiss Zen 2012 (Blue edition). Representative wide-field fluorescence images cropped from ImageJ counter-Window images acquired using an EC Plan-Neofluar 10× /0.30 Ph1 lens demonstrating the distance of Lyve-1 + macrophages (green) to the lymphatic vessel walls (green with Prox-1 in purple) in PFA-fixed E15.5 dorsal skin whole-mounts of WT, ACKR2-KO and CCR2-KO embryos. A graph showing the mean distance of Lyve-1 + macrophages to the lymphatic vessel walls of PFA-fixed E15.5 dorsal skin whole-mounts. Each point on the graph represents data from a single embryo. One-way ANOVA was used for statistical analysis with differences between groups analysed by a post-test using Newman–Keul multiple comparison test as a post-test.

Article Snippet: Forelimb skin or dorsal skin was isolated using fine forceps and scissors (Fine Science Tools, Germany) under a dissection microscope and incubated with 2 μg/ml anti-VEGFR3/anti-Lyve-1 or 1:2,000 anti-Prox-1 antibodies in cRPMI/0.05% Triton X-100 (Sigma-Aldrich, UK) overnight at 4°C with gentle agitation.

Techniques: Fluorescence, Staining, Generated

Journal: Regenerative engineering and translational medicine

Article Title: Cooperative Effects of Vascular Angiogenesis and Lymphangiogenesis

doi: 10.1007/s40883-018-0054-2

Figure Lengend Snippet: (A) Lymphatic endothelial cells (LECs) were adhered to microchannels in 12 h, resulting in the formation of the lymphatic vessel in 24–36h. (B) Lymphatic morphogenesis was induced in a dose-dependent manner of VEGF-C (10, 50 ng/ml) at D3, D5, and D7. Podoplanin was expressed in lymphatic angiogenic sprouting. (C) Lumen structure of lymphatic vessel after 10 days of perfusion culture. (D, E) Lymphatic sprouting and single cell migration stained by podoplanin and Prox-1. (F) Relative gene expression changes at D1 and D10 (VEGF-C 10 and 50 ng/ml). High concentration of VEGF-C induces increasing the expression of VEGF-R3, Prox1, and ORAI1. *, p<0.01, error bar=±SD.

Article Snippet: Cells were then incubated with the PE-conjugated anti-human podoplanin antibody (Biolegend, 1:200) and anti-Prox-1 antibody (Abcam, 1:100) and anti-VE-cadherin (CD144) antibody (Biolegend) for 2 h at room temperature.

Techniques: Migration, Staining, Expressing, Concentration Assay