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Journal of Visualized Experiments: JoVE

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https://www.readbyqxmd.com/read/29155775/mapping-genome-wide-accessible-chromatin-in-primary-human-t-lymphocytes-by-atac-seq
#1
Ivana Grbesa, Miriam Tannenbaum, Avital Sarusi-Portuguez, Michal Schwartz, Ofir Hakim
Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions of chromatin. These regions represent regulatory DNA elements (e.g., promoters, enhancers, locus control regions, insulators) to which transcription factors bind. Mapping the accessible chromatin landscape is a powerful approach for uncovering active regulatory elements across the genome. This information serves as an unbiased approach for discovering the network of relevant transcription factors and mechanisms of chromatin structure that govern gene expression programs...
November 13, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29155771/endotracheal-intubation-of-rabbits-using-a-polypropylene-guide-catheter
#2
Krista L Thompson, Thomas R Meier, Jodi A Scholz
Endotracheal intubation in rabbits can be challenging due to their unusual anatomy. Achieving a patent airway during anesthesia is critical for the avoidance of airway obstruction, prevention of gastric tympany, and to allow ventilatory support. Due to the difficulty of intubation, alternative methods such as the use of laryngeal mask airways or laryngeal tubes have been explored. However, these methods do not result in direct access to the trachea and thus may present a risk for development of complications...
November 13, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29155765/continuous-hydrologic-and-water-quality-monitoring-of-vernal-ponds
#3
Odette Mina, Heather E Gall, Joseph W Chandler, Jeremy Harper, Malcolm Taylor
Vernal ponds, also referred to as vernal pools, provide critical ecosystem services and habitat for a variety of threatened and endangered species. However, they are vulnerable parts of the landscapes that are often poorly understood and understudied. Land use and management practices, as well as climate change are thought to be a contribution to the global amphibian decline. However, more research is needed to understand the extent of these impacts. Here, we present methodology for characterizing a vernal pond's morphology and detail a monitoring station that can be used to collect water quantity and quality data over the duration of a vernal pond's hydroperiod...
November 13, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29155743/snap-chip-for-cross-reactivity-free-and-spotter-free-multiplexed-sandwich-immunoassays
#4
Huiyan Li, Sebastien Bergeron, Heidi Larkin, David Juncker
Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays...
November 13, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29155761/advanced-confocal-microscopy-techniques-to-study-protein-protein-interactions-and-kinetics-at-dna-lesions
#5
Soňa Legartová, Jana Suchánková, Jana Krejčí, Alena Kovaříková, Eva Bártová
Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented...
November 12, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29155757/dissection-of-human-retina-and-rpe-choroid-for-proteomic-analysis
#6
Thiago Cabral, Marcus A Toral, Gabriel Velez, James E DiCarlo, Anuradha M Gore, MaryAnn Mahajan, Stephen H Tsang, Alexander G Bassuk, Vinit B Mahajan
The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular choroid layer. Different regions of the retina are anatomically and molecularly distinct, facilitating unique functions and demonstrating differential susceptibility to disease. Proteomic analysis of each of these regions and layers can provide vital insights into the molecular process of many diseases, including Age-Related Macular Degeneration (AMD), diabetes mellitus, and glaucoma...
November 12, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29155795/in-vivo-imaging-of-cx3cr1gfp-gfp-reporter-mice-with-spectral-domain-optical-coherence-tomography-and-scanning-laser-ophthalmoscopy
#7
Despina Kokona, Joël Jovanovic, Andreas Ebneter, Martin S Zinkernagel
Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1(gfp/gfp)) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases(1)(,)(2)(,)(3)(,)(4)(,)(5)(,)(6). This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1(gfp/gfp) mice with SLO in vivo, using an ophthalmic imaging platform system...
November 11, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29155768/slice-patch-clamp-technique-for-analyzing-learning-induced-plasticity
#8
Hiroyuki Kida, Yuya Sakimoto, Dai Mitsushima
The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze motor-learning induced plasticity, we trained rats using an accelerated rotor rod task. Rats performed the task 10 times at 30-s intervals for 1 or 2 days. Performance was significantly improved on the training days compared to the first trial. We then prepared acute brain slices of the primary motor cortex (M1) in untrained and trained rats. Current-clamp analysis showed dynamic changes in resting membrane potential, spike threshold, afterhyperpolarization, and membrane resistance in layer II/III pyramidal neurons...
November 11, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29155787/synthesis-of-core-shell-lanthanide-doped-upconversion-nanocrystals-for-cellular-applications
#9
Xiangzhao Ai, Linna Lyu, Jing Mu, Ming Hu, Zhimin Wang, Bengang Xing
Lanthanide-doped upconversion nanocrystals (UCNs) have attracted much attention in recent years based on their promising and controllable optical properties, which allow for the absorption of near-infrared (NIR) light and can subsequently convert it into multiplexed emissions that span over a broad range of regions from the UV to the visible to the NIR. This article presents detailed experimental procedures for high-temperature co-precipitation synthesis of core-shell UCNs that incorporate different lanthanide ions into nanocrystals for efficiently converting deep-tissue penetrable NIR excitation (808 nm) into a strong blue emission at 480 nm...
November 10, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29155784/correlative-super-resolution-and-electron-microscopy-to-resolve-protein-localization-in-zebrafish-retina
#10
José M Mateos, Gery Barmettler, Jana Doehner, Irene Ojeda Naharros, Bruno Guhl, Stephan C F Neuhauss, Andres Kaech, Ruxandra Bachmann-Gagescu, Urs Ziegler
We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM)...
November 10, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29155776/differential-effects-of-lipid-lowering-drugs-in-modulating-morphology-of-cholesterol-particles
#11
Shanmugavel Madasamy, David Liu, Jason Lundry, Benjamin Alderete, Raymond Kong, J Paul Robinson, Alan H B Wu, Edward P Amento
Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to moderate level of increase in high-density lipoprotein (HDL) cholesterol in plasma. However, a possible role of these drugs in altering morphology and distribution of cholesterol particles is poorly understood. Here, we describe the in vitro evaluation of lipid-lowering drug effects in modulating morphological features of cholesterol particles using the plaque array method in combination with imaging flow cytometry...
November 10, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29155772/preparation-of-primary-acute-lymphoblastic-leukemia-cells-in-different-cell-cycle-phases-by-centrifugal-elutriation
#12
Magdalena Delgado, Anisha Kothari, Walter N Hittelman, Timothy C Chambers
The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum deprivation; chemicals which arrest cells at different cell cycle phases; or the use of mitotic shake-off which exploits their reduced adherence. However, all of these have disadvantages. For example, serum starvation works well for normal cells but less well for tumor cells with compromised cell cycle checkpoints due to oncogene activation or tumor suppressor loss...
November 10, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29155792/assessment-of-dictyostelium-discoideum-response-to-acute-mechanical-stimulation
#13
Yulia Artemenko, Peter N Devreotes
Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known...
November 9, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29155773/dna-magnetic-particle-binding-analysis-by-dynamic-and-electrophoretic-light-scattering
#14
Yazan Haddad, Simona Dostalova, Jiri Kudr, Ondrej Zitka, Zbynek Heger, Vojtech Adam
Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA...
November 9, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29155769/a-simple-approach-to-induce-experimental-autoimmune-neuritis-in-c57bl-6-mice-for-functional-and-neuropathological-assessments
#15
David G Gonsalvez, Jessica L Fletcher, Sang Won Yoo, Rhiannon J Wood, Simon S Murray, Junhua Xiao
Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by immunizing mice with neurogenic peptides to direct an inflammatory attack toward components of the peripheral nervous system (PNS). Recent advances have enabled the induction of EAN in the relatively resistant C57BL/6 mouse line using myelin protein zero (P0)106-125 or P0180-199 peptides delivered in adjuvant combined with the injection of pertussis toxin...
November 9, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29155764/multiplex-cytokine-profiling-of-stimulated-mouse-splenocytes-using-a-cytometric-bead-based-immunoassay-platform
#16
Jason S Lehmann, Amy Zhao, Binggang Sun, Weiping Jiang, Shaoquan Ji
Bead-based immunoassays employ the same basic principle as sandwich immunoassays. Capture beads, which can be differentiated by size and internal allophycocyanin (APC) fluorescence intensity, are conjugated to antibodies specific to a particular analyte. Next, a selected panel of defined capture bead sets is incubated with a biological sample containing target analytes specific to the capture antibodies. A biotinylated detection antibody cocktail is added, which leads to the formation of capture bead-analyte-detection antibody sandwiches...
November 9, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29155794/an-array-based-comparative-genomic-hybridization-platform-for-efficient-detection-of-copy-number-variations-in-fast-neutron-induced-medicago-truncatula-mutants
#17
Yuhui Chen, Xianfu Wang, Shunfei Lu, Hongcheng Wang, Shibo Li, Rujin Chen
Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including biological such as T-DNA or transposon, chemical such as ethyl methanesulfonate (EMS), or physical such as ionization radiation. The type of mutation observed varies depending on the mutagen used. For ionization radiation induced mutants, mutations include deletion, duplication, or rearrangement. While T-DNA or transposon-based mutagenesis is limited to species that are susceptible to transformation, chemical or physical mutagenesis can be applied to a broad range of species...
November 8, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29155789/efficient-generation-and-editing-of-feeder-free-ipscs-from-human-pancreatic-cells-using-the-crispr-cas9-system
#18
Anjali Nandal, Barbara Mallon, Bhanu P Telugu
Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few...
November 8, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29155766/assessment-of-blood-brain-barrier-permeability-by-intravenous-infusion-of-fitc-labeled-albumin-in-a-mouse-model-of-neurodegenerative-disease
#19
Alba Di Pardo, Salvatore Castaldo, Luca Capocci, Enrico Amico, Maglione Vittorio
Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies. Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease...
November 8, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29155740/creation-of-a-rodent-model-of-abdominal-aortic-aneurysm-by-blocking-adventitial-vasa-vasorum-perfusion
#20
Hiroki Tanaka, Naoki Unno, Tatsuro Yata, Hirona Kugo, Nobuhiro Zaima, Takeshi Sasaki, Tetsumei Urano
The adventitial vasa vasorum (VV) provides oxygen and nourishment to the aortic wall. Hypoxia in the aortic wall can cause enlarged abdominal aortic aneurysms (AAAs). This article introduces and describes a standard protocol used to induce AAAs through adventitial VV hypoperfusion created with a combination of polyurethane catheter insertion into the aortic lumen and suture ligation of the infrarenal abdominal aorta. The protocol involves the use of male rats weighing 300-400 g, which are provided food and water ad libitum...
November 8, 2017: Journal of Visualized Experiments: JoVE
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