cd31 a488  (R&D Systems)

Journal: Nature Communications

Article Title: Longitudinal intravital imaging of the femoral bone marrow reveals plasticity within marrow vasculature

doi: 10.1038/s41467-017-01538-9

Figure Lengend Snippet: The bone marrow within the imaging volume reaches steady-state comparable to homeostasis 28 days after LIMB implantation. a Immunofluorescence analysis of bone sections after removal of the LIMB implant over the time course of 4 weeks. ECM formation was identified by the marker Laminin (Lam). Stem-cell antigen 1 (Sca-1) is highly expressed in arterioles. The leukocyte marker CD45 indicates localization of inflammatory cells adjacent to the window cavity (wc). Lam is highly expressed around the implant during the first week and completely normalizes after 2–4 weeks. CD45 + cell accumulations are found during the first weeks in proximity to the wc. b Movat’s pentachrome stain detects connective tissues and reveals remodeling of bone primarily on the periosteal interface near the fixation plate. a , b Images are representative for 3–5 mice per time point post-surgery. c Overview immunofluorescence images of the femoral bones from an individual mouse 3 days post-surgery. Note the specific reaction to the implant-bone marrow interfaces indicated by accumulations of CD45 + cells, and Lam + and Sca1 + arteries (yellow). bm bone marrow, cb cortical bone. d Immunofluorescence image of the region around the endoscope tubing in a LIMB-implanted femur 7 days post-surgery, including the bone cortex and periosteum under the plate. The presence of various blood vessel subsets indicated by CD31 and Emcn demonstrates intact blood supply to the periosteum and to the bone. Scale bar = 100 µm. e Blood supply is intact throughout the marrow cavity, indicated by CMTPX-labeled splenocytes, which localize in the bone marrow 4 h after transplantation in both contralateral and LIMB-implanted femurs at 42 days post-surgery ( n = 3 mice). f Histological DAPI stain (gray) shows intact tissue structure, with no separation of the bone marrow and the vasculature by the screws or endoscope tubing. g Flow cytometry analysis of femurs with the LIMB implant, their contralateral femurs and femurs of control mice. Similar frequencies and cell counts of various cell populations shows no effect of the LIMB implant on bone marrow cell composition ( n = 8 LIMB-implanted mice, n = 8 controls, two independent experiments). Error bars represent s.e.m. values. Statistical analysis was performed using t -test

Article Snippet: Sections of 7 μm were blocked with 5% FCS/PBS for 30 min and stained with antibodies in 5% FCS/PBS/0.1% Tween for 1–2 h at room temperature: CD45 (1:100, ThermoFisher eBioscience, Frankfurt, Germany, 30-F11), Sca-1-APC (1:200, eBioscience, 17-5981-82), Laminin (1:200, Sigma-Aldrich, Taufkirchen, Germany, L9393), c-kit-PE (1:100, 130-102-542, Miltenyi, Bergisch Gladbach, Germany), Ki-67-bio (1:100, eBioscience, 14-5698-82), Endomucin (1:100, Santa Cruz, sc-65495), CD31-A488 (1:100, R&D Systems, FAB3628G).

Techniques: Imaging, Immunofluorescence, Marker, Staining, Labeling, Transplantation Assay, Flow Cytometry, Control

Journal: Nature Communications

Article Title: Longitudinal intravital imaging of the femoral bone marrow reveals plasticity within marrow vasculature

doi: 10.1038/s41467-017-01538-9

Figure Lengend Snippet: LIMB and immunofluorescence analysis indicate possible mechanisms of vascular morphological changes deep in the femoral bone marrow, during regeneration, and in steady-state homeostasis. a Immunofluorescence analysis shows that type H vessels, characterized by CD31 hi Emcn hi -expressing endothelial cells, are induced and present around the implant at day 3 after LIMB implantation. Their presence may vary individually but normalizes within 28 days post-surgery. Sinusoidal and type H vessel morphology adjacent to the wc is irregular in the first week and completely reorganizes to an appearance comparable to vessels found at endosteal areas distant from the injury site ( n = 3 mice). bm bone marrow, cb cortical bone. Scale bar = 500 µm (left panels). b Immunofluorescence analysis after EdU pulse-chase experiments indicates similar EdU-uptake in the bone marrow of LIMB-implanted femurs and contralateral bones. Proliferating endothelial cells were rarely present at late time points after implantation. This result also supports the conclusion that 28 days after LIMB implantation both the bone and the bone marrow reach homeostasis ( n = 3 mice in each cohort). c 3D fluorescence image (300 × 300 × 66 µm 3 , left and right panel) acquired by LIMB 26 days post-surgery, in a paGFP mouse with the vasculature labeled by Qdots. Photoactivation was performed within a volume of 100 × 100 × 9 µm 3 in the center of the image. The fluorescence image was acquired 2 h post activation. Scale bar = 50 µm. The middle panel shows time-lapse 3D images of the inset from the left panel, indicating that paGFP fluorescent cells outside the initial photoactivation volume are present 3 h after photoactivation and that they fluctuate in number and position within the tissue. Passive displacement of the relatively immobile stromal and vascular compartments by continuous proliferation and movement of hematopoietic cells is a possible mechanism of tissue and vascular re-localization during homeostasis (see Supplementary Movies , )

Article Snippet: Sections of 7 μm were blocked with 5% FCS/PBS for 30 min and stained with antibodies in 5% FCS/PBS/0.1% Tween for 1–2 h at room temperature: CD45 (1:100, ThermoFisher eBioscience, Frankfurt, Germany, 30-F11), Sca-1-APC (1:200, eBioscience, 17-5981-82), Laminin (1:200, Sigma-Aldrich, Taufkirchen, Germany, L9393), c-kit-PE (1:100, 130-102-542, Miltenyi, Bergisch Gladbach, Germany), Ki-67-bio (1:100, eBioscience, 14-5698-82), Endomucin (1:100, Santa Cruz, sc-65495), CD31-A488 (1:100, R&D Systems, FAB3628G).

Techniques: Immunofluorescence, Expressing, Pulse Chase, Fluorescence, Labeling, Activation Assay