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