Computational protocol: Role of CX3CR1 Receptor in Monocyte/Macrophage Driven Neovascularization

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Protocol publication

[…] 5 µM thick frozen sections from carotid artery were cut and stained with hematoxylin-Z and eosin and Trichrome stains (CellPath Ltd, Powys, UK). Tissue images were acquired using Nikon CCD camera attached to a Nikon microscope. Morphometric analysis was performed using NIH ImageJ software for data quantification.For immunofluorescence detection of smooth muscle specific protein and GFP expression carotid artery/Matrigel sections were stained for calponin (rabbit anti calponin: 1∶200; Epitomics, CA, USA), CX3CR1 (rabbit anti CX3CR1:1∶50; ProSci Incorporated, CA, USA), F4/80 (Rat anti mouse F4/80: 1∶100; eBioscience; Hatfield, UK), Laminin (mouse anti laminin: 1∶500; Novus Biologicals, Cambridge, UK) and/or CD42b (rat anti CD42b: 1∶200; emfret Analytics GmbH & co.KG, Eibelstadt, Germany). Goat anti-rabbit, anti-mouse or anti rat Alexa Fluor 546 or 488 (1∶250 or 500; Abcam, Cambridge, UK) was used as secondary antibody and DAPI was used for nuclear staining. Rabbit, mouse or rat IgG (1∶50 or 200; Abcam, Cambridge, UK) were used as isotype controls. No additional labelling was required for GFP. Several thin optical slices (thickness<0.7 µm) of high-resolution sequential confocal scans (Nikon eC1 plus, TE2000E) of carotid artery sections were acquired to identify single GFP+ cells co-expressing smooth muscle marker and to avoid false positive images due to overlapping cells. The acquired images were coded and analysed by two observers blinded to the codes. The 3D reconstruction and analysis of the images was performed using IMARIS software (Bitplane Scientific Software, Zurich, Switzerland). […]

Pipeline specifications

Software tools ImageJ, Imaris
Application Microscopic phenotype analysis
Organisms Mus musculus
Diseases Atherosclerosis