1Department of Neuroscience, IRCCS - Istituto di Ricerche Farmacologiche Mario Negri
A way to gain new insights into the complexity of the brain inflammatory response is presented. We describe immunofluorescence-based protocols followed by three-dimensional confocal analysis to investigate the pattern of co-expression of microglia/macrophage phenotype markers in a mouse model of focal ischemia.
Published September 4, 2013. Keywords: Neurobiology, Neuroscience, Molecular Biology, Cellular Biology, Medicine, Biomedical Engineering, Anatomy, Physiology, Central Nervous System Diseases, Neurodegenerative Diseases, biology (general), immunology, life sciences, animal models, Inflammation, stroke, alternative activation, brain injury, brain, imaging, confocal microscopy, three-dimensional imaging, clinical techniques, mouse, animal model
1Cell and Developmental Biology, University of Michigan, 2Department of Biosciences and Nutrition, Karolinska Instituet Novum
Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.
Published September 4, 2014. Keywords: Molecular Biology, Developmental Biology, morphogenesis, mouse fetal intestine, whole organ culture, live imaging, cell signaling, three-dimensional reconstruction, two-photon imaging
1Institute of Immunology & Experimental Oncology, Center for Biomedical Education and Research (ZBAF), Witten/Herdecke University
Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.
Published October 5, 2014. Keywords: Bioengineering, cell migration, 3D collagen matrix, cell tracking
1Department of Mechanical and Aerospace Engineering, The George Washington University, 2Department of Mechanical and Aeronautical Engineering, Clarkson University
Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient's ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.
Published February 3, 2014. Keywords: Bioengineering, oil-flow visualization, vocal fold polyp, three-dimensional flow separation, aerodynamic pressure loadings
1Team Alzheimer & Tauopathies, Jean-Pierre Aubert Research Centre, Inserm UMR 837, 2EA 4308-Department of Reproductive Biology-Spermiology-CECOS, CHRU-Lille, 3EA2686-Laboratorie d'Immunologie, Faculté de Médecine - Pôle Recherche, 4Department of Neurology, CHRU-Lille
A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.
Published April 10, 2014. Keywords: Neuroscience, proteomics, neurodegeneration, 2DE, human and mice brain tissue, fluorescence, immunoblotting.
Abbreviations: 2DE (two-dimensional gel electrophoresis), 2D-DIGE (two-dimensional fluorescence difference gel electrophoresis), mini-2DE (mini 2DE immunoblotting), IPG (Immobilized pH Gradients), IEF (isoelectrofocusing), AD (Alzheimer´s disease)
1Department of Physics and Astronomy, University of Maine
We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers within single cells.
Published December 9, 2013. Keywords: Basic Protocol, Microscopy, Super-resolution imaging, Multicolor, single molecule, FPALM, Localization microscopy, fluorescent proteins
1New Jersey Neuroscience Institute, JFK Medical Center, 2Department of Biomedical Engineering, Rutgers University, 3Department of Mechanical and Aerospace Engineering, Rutgers University
Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.
Published August 27, 2014. Keywords: Bioengineering, bioengineering (general), Elastic, viscoelastic, bis-acrylamide, substrate, stiffness, dynamic, static, neuron, fibroblast, compliance, ECM, mechanobiology, tunable
JoVE Clinical and Translational Medicine
1Department of Internal Medicine, University of Michigan Comprehensive Cancer Center, 2Department of Pathology, University of Michigan Comprehensive Cancer Center
Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.
Published April 25, 2014. Keywords: Medicine, pathological conditions, signs and symptoms, neoplasms, three dimensional cultures, Matrigel, breast cells, malignant phenotype, signaling
1The Rowland Institute, Harvard University, 2Faculdade de Ciências e Letras de Assis, Universidade Estadual Paulista
The three-dimensional locations of weakly-scattering objects can be uniquely identified using digital inline holographic microscopy (DIHM), which involves a minor modification to a standard microscope. Our software uses a simple imaging heuristic coupled with Rayleigh-Sommerfeld back-propagation to yield the three-dimensional position and geometry of a microscopic phase object.
Published February 8, 2014. Keywords: Basic Protocol, holography, digital inline holographic microscopy (DIHM), Microbiology, microscopy, 3D imaging, Streptococcus bacteria
JoVE Application Notes
In vitro mammalian cell culture has served as an invaluable tool in cell biology for several decades. Classically, monolayer cultures of adherent cells were grown on flat and rigid two-dimensional (2D) substrates, such as polystyrene or glass. However, many cells, when isolated from tissues and placed onto stiff planar 2D cell culture surfaces, such as tissue culture plastic, become progressively flatter, divide aberrantly, and lose their differentiated phenotype1,2. While these two-dimensional cell culture studies have played a pivotal role in furthering our understanding of many biological processes, they do not emulate in vivo conditions.
Published October 16, 2014. Keywords: