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Journal of Visualized Experiments: JoVE

Bhavya Ravi, Layla M Nassar, Richard J Kopchock, Pravat Dhakal, Michael Scheetz, Kevin M Collins
It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified...
February 7, 2018: Journal of Visualized Experiments: JoVE
Elizabeth M Corteselli, James M Samet, Eugene A Gibbs-Flournoy
While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses...
February 7, 2018: Journal of Visualized Experiments: JoVE
Melva Suarez Muñoz, Davide Confalonieri, Heike Walles, Elisabeth M W M van Dongen, Gudrun Dandekar
Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods...
February 7, 2018: Journal of Visualized Experiments: JoVE
Grigol Tediashvili, Dong Wang, Hermann Reichenspurner, Tobias Deuse, Sonja Schrepfer
The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely...
February 7, 2018: Journal of Visualized Experiments: JoVE
Charlotte A Scarff, Martin J G Fuller, Rebecca F Thompson, Matthew G Iadaza
Negative stain electron microscopy (EM) allows relatively simple and quick observation of macromolecules and macromolecular complexes through the use of contrast enhancing stain reagent. Although limited in resolution to a maximum of ~18 - 20 Å, negative stain EM is useful for a variety of biological problems and also provides a rapid means of assessing samples for cryo-electron microscopy (cryo-EM). The negative stain workflow is straightforward method; the sample is adsorbed onto a substrate, then a stain is applied, blotted, and dried to produce a thin layer of electron dense stain in which the particles are embedded...
February 6, 2018: Journal of Visualized Experiments: JoVE
Sandra B Lemke, Frank Schnorrer
Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment...
February 6, 2018: Journal of Visualized Experiments: JoVE
Chaoran Meng, Zhaoling Li, Chaoyun Wang, Chongwen Yu, Xuerong Bi, Siyi Wang
This protocol demonstrates a method for ramie fiber extraction by scouring raw ramie in an alkali hydrogen peroxide system supported by a controlled-release alkali source. The fiber extracted from ramie is a type of textile material of great importance. In previous studies, ramie fiber was extracted in an alkali hydrogen peroxide system supported only by sodium hydroxide.However, due to the strong alkalinity of sodium hydroxide, the oxidation reaction speed of hydrogen peroxide was difficult to control and thus resulted in great damage to the treated fiber...
February 6, 2018: Journal of Visualized Experiments: JoVE
Marie-Claire Devilder, Mélinda Moyon, Xavier Saulquin, Laetitia Gautreau-Rolland
Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes...
February 6, 2018: Journal of Visualized Experiments: JoVE
Brandon M Wild, Renée Morris, Mihai Moldovan, Christian Krarup, Arun V Krishnan, Ria Arnold
Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and latency detect chronic axon loss and demyelination, respectively. Axonal excitability techniques "by threshold tracking" expand upon these measures by providing information regarding the activity of ion channels, pumps and exchangers that relate to acute function and may precede degenerative events. As such, the use of axonal excitability in animal models of neurological disorders may provide a useful in vivo measure to assess novel therapeutic interventions...
February 6, 2018: Journal of Visualized Experiments: JoVE
Sang A Park, Young Ho Choe, Sung Hwan Lee, Young-Min Hyun
Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs)...
February 6, 2018: Journal of Visualized Experiments: JoVE
Stephen R Wellard, Jessica Hopkins, Philip W Jordan
Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during meiosis can lead to infertility, pregnancy loss or genetic defects. Commencing at the onset of puberty and continuing throughout adulthood, continuous semi-synchronous waves of spermatocytes undergo spermatogenesis and ultimately form haploid sperm. The first wave of mouse spermatocytes undergoing meiotic initiation appear at day 10 post-partum (10 dpp) and are released into the lumen of seminiferous tubules as mature sperm at 35 dpp...
February 6, 2018: Journal of Visualized Experiments: JoVE
Shelby Shrigley, Karolina Pircs, Roger A Barker, Malin Parmar, Janelle Drouin-Ouellet
Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process...
February 5, 2018: Journal of Visualized Experiments: JoVE
Erica L McGrath, Junling Gao, Ping Wu
Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental neurobiology. Rather than use an animal model or genetically modified induced pluripotent cells, human neural stem cells provide an effective in vitro system to examine the effects of treatments, screen drugs, or examine individual differences. Here, we provide the detailed protocols for methods used to expand human fetal brain neural stem cells in culture with serum-free media, to differentiate them into various neuronal subtypes and astrocytes via different priming procedures, and to freeze and recover these cells...
February 5, 2018: Journal of Visualized Experiments: JoVE
Chiara Molinari, Raefa Abou Khouzam, Samanta Salvi, Tania Rossi, Guglielmina Nadia Ranzani, Daniele Calistri
CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields...
February 5, 2018: Journal of Visualized Experiments: JoVE
Youkyeong Gloria Byun, Won-Suk Chung
Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major immune cells and only phagocytes in the brain, recent studies have shown that astrocytes also participate in various phagocytic processes, such as developmental synapse elimination and clearance of amyloid beta plaques in Alzheimer's disease (AD). Despite these findings, the efficiency of astrocyte engulfment and degradation of their targets is unclear compared with that of microglia...
February 5, 2018: Journal of Visualized Experiments: JoVE
Morgan Philp, Ronald Shimmon, Mark Tahtouh, Shanlin Fu
Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and other border protection agencies globally. Color testing is a presumptive identification technique indicating the presence or absence of a particular drug class using rapid and uncomplicated chemical methods. Owing to their relatively recent emergence, a color test for the specific identification of synthetic cathinones is not currently available. In this study, we introduce a protocol for the presumptive identification of synthetic cathinones, employing three aqueous reagent solutions: copper(II) nitrate, 2,9-dimethyl-1,10-phenanthroline (neocuproine) and sodium acetate...
February 5, 2018: Journal of Visualized Experiments: JoVE
Sachin S Shivatare, Vidya S Shivatare, Chung-Yi Wu, Chi-Huey Wong
We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are commonly found on human glycoproteins. To achieve the desired structural diversity, the strategy began with the chemo-enzymatic synthesis of three kinds of oligosaccharyl fluoride modules, followed by their stepwise α-selective glycosylations at the 3-O and 6-O positions of the mannose residue of the common core trisaccharide having a crucial β-mannoside linkage...
February 5, 2018: Journal of Visualized Experiments: JoVE
Aurelia C Kucera, Britt J Heidinger
Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. Birds are widely used subjects in biological research and are ideal for studies involving repeated sperm samples. However, few resources are currently available for those wishing to learn how to collect and extract DNA from avian sperm. Here we describe cloacal massage, a gentle, non-invasive manual technique for collecting avian sperm. Although this technique is established in the literature, it can be difficult to learn from the available descriptions...
February 5, 2018: Journal of Visualized Experiments: JoVE
Andrea Brigitta Alber, David Michael Suter
Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used approaches to determine protein half-life are either limited to population averages from lysed cells or require the use of protein synthesis inhibitors. This protocol describes a method to measure protein half-lives in single living adherent cells, using SNAP-tag fusion proteins in combination with fluorescence time-lapse microscopy. Any protein of interest fused to a SNAP-tag can be covalently bound by a fluorescent, cell permeable dye that is coupled to a benzylguanine derivative, and the decay of the labeled protein population can be monitored after washout of the residual dye...
February 4, 2018: Journal of Visualized Experiments: JoVE
Hasan Goktas
Here, we demonstrate the advantages of the laser Doppler vibrometer (LDV) over conventional techniques (the network analyzer), as well as the techniques to create an application-based microelectromechanical systems (MEMS) filter and how to use it efficiently (i.e., tuning the tuning-capability and avoiding both failure and stiction). LDV enables crucial measurements that are impossible with the network analyzer, such as higher mode detection (highly sensitive biosensor application) and resonance measurement for very small devices (fast prototyping)...
February 4, 2018: Journal of Visualized Experiments: JoVE
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