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Biochemistry

Quantification of Bacterial Fatty Acids by Extraction and Methylation Authors:  Mark Politz, Rebecca Lennen and Brian Pfleger, date: 11/05/2013, view: 7785, Q&A: 1
This protocol describes two similar methods for the extraction and methylation of fatty acids from bacterial cultures. The acid derivatization protocol (Lennen et al., 2013; Bligh and Dyer, 1959) results in the extraction and methylation of all fatty acids, both free and bound, from a bacterial culture, while the base derivatization ...
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β1 Integrin Cell-surface Immunoprecipitation (Selective Immunoprecipitation) Author:  Ralph T. Böttcher, date: 11/05/2013, view: 5565, Q&A: 0
Immunoprecipitation (IP) is a widely used method to isolate a specific protein from a mixed protein sample using an antibody that exclusively binds to that particular protein. This technique allows studying protein-protein and protein-nucleic acid interactions or to identify post-translational protein modifications. Many proteins, in particular ...
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Cell Biology

Ki67 Immunofluorescence on Bovine Cell Lines Authors:  Justine Marsolier, Souhila Medjkane, Martine Perichon and Jonathan B. Weitzman, date: 11/05/2013, view: 4296, Q&A: 0
This is a rapid protocol to test the effects of drugs treatment on bovine cell replication using Ki67 staining. Ki67 is associated with cell proliferation and is present during all active phases of the cell cycle (G1, S, G2, and mitosis), but is absent from resting cells (G0).
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Immunology

Bacterial Counts in Spleen Authors:  Elías Barquero-Calvo, Carlos Chacón-Díaz, Esteban Chaves-Olarte and Edgardo Moreno, date: 11/05/2013, view: 12302, Q&A: 0
Bacterial loads can be determined as colony forming units (CFU) at any point of the infection by culturing spleen homogenates on agar plates. This is a reliable method for comparing the kinetics of infection in various mouse strains, estimating the virulence of different bacterial mutants or isolates and for vaccine testing and vacine ...
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Microbiology

H2O2 Kill Assays of Biofilm Bacteria Authors:  Malika Khakimova and Dao Nguyen, date: 11/05/2013, view: 5373, Q&A: 0
Ubiquitous in nature and often surface associated, biofilms cause numerous chronic human infections. Biofilms are structured multicellular bacterial communities where cells are entrapped in a polymer matrix. Bacteria growing as biofilms are characterized by marked tolerance to many biocides, including oxidants such as hydrogen peroxide. Hydrogen ...
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H2O2 Kill Assays of Planktonic Stationary Phase Bacteria Authors:  Malika Khakimova and Dao Nguyen, date: 11/05/2013, view: 4935, Q&A: 0
Stationary phase bacteria are highly tolerant to hydrogen peroxide. This protocol was developed to test the susceptibility to hydrogen peroxide killing in different Pseudomonas aeruginosa strains. This assay provides a reliable way to measure killing of stationary phase bacterial cells to hydrogen peroxide and can be adapted to test other ...
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Adhesion of Moraxella catarrhalis to Respiratory Tract Epithelial Cells Authors:  Stefan P.W. de Vries and Hester J. Bootsma, date: 11/05/2013, view: 5056, Q&A: 0
Moraxella catarrhalis is a human-restricted pathogen that is responsible for respiratory tract infections such as childhood otitis media (OM) and exacerbation of chronic obstructive pulmonary disease (COPD) in adults. Successful colonization and infection by M. catarrhalis depends on its ability to attach to the respiratory tract ...
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Analysis of Moraxella catarrhalis Outer Membrane Protein Profiles Authors:  Stefan P.W. de Vries and Hester J. Bootsma, date: 11/05/2013, view: 4451, Q&A: 0
Phenotypes observed for certain Moraxella catarrhalis wild-type strains or mutants may be caused by a variety of factors including alteration of outer membrane protein composition. Examination of the outer membrane protein profiles may be a valuable tool to identify changes in outer membrane compositions of these strains. Here we describe ...
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Immunoplaque Assay (Influenza Virus) Authors:  Longping V. Tse, Yueting Zhang and Gary R. Whittaker, date: 11/05/2013, view: 10457, Q&A: 0
Despite developed long time ago, plaque assay is still the gold standard for viral titer quantification in modern virology. The standard crystal violet-based plaque assay relies on virus’ ability to induce cytopathic effect (CPE) which limits the assay to lytic viruses. Alternative viral quantification assays such as 50% tissue culture infectious ...
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Neuroscience

Dissection of Different Areas from Mouse Hippocampus Author:  Faraz A. Sultan, date: 11/05/2013, view: 10856, Q&A: 0
The hippocampus modulates a number of modules including memory consolidation, spatial navigation, temporal processing and emotion. A banana-shaped structure, the hippocampus is constituted of morphologically distinct subregions including the dentate gyrus, CA3 and CA1 (here, we do not distinguish the “hippocampus proper” which consists only of ...
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Mitochondrial Isolation and Purification from Mouse Spinal Cord Authors:  Philippe A. Parone, Sandrine Da Cruz and Don W. Cleveland, date: 11/05/2013, view: 5231, Q&A: 0
Mitochondria are eukaryotic organelles that play a crucial role in several cellular processes, including energy production, β-oxidation of fatty acids and regulation of calcium homeostasis. In the last 20 years there has been a hightened interest in the study of mitochondria following the discoveries that mitochondria are central to the process of ...
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Implantation of Dkk-1-soaked Beads into the Neural Tube of Chicken Embryos Authors:  Almudena Martinez-Ferre, Maria Navarro-Garberí, Carlos Bueno and Salvador Martinez, date: 11/05/2013, view: 6408, Q&A: 0
Chick embryos are known to be a powerful system to test gene function due to the “in vivo” accessibility, short time for results retrieval and the possibility to perform a large number of experiments. Synthetic micro-beads soaked in morphogenetic signals or receptor inhibitors can be implanted in selective embryo regions at precise ...
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NMDA-induced Excitotoxicity and Lactate Dehydrogenase Assay in Primary Cultured Neurons Authors:  Shu Zhang and Yu Tian Wang, date: 11/05/2013, view: 6674, Q&A: 0
N-Methyl-D-aspartic acid receptor (NMDAR)-mediated excitotoxicity is thought to contribute to the pathogenesis of a large number of chronic neurodegenerative disorders (such as Alzheimer’s and Huntington’s diseases to mental illnesses) in addition to acute brain insults such as stroke and brain trauma. Understanding the mechanisms underlying ...
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Plant Science

Ovule Clearing Method for Solanaceous Species Authors:  Audrey Loubert-Hudon and Daniel P. Matton, date: 11/05/2013, view: 4993, Q&A: 0
Depending on the species, embryo sacs can be difficult to observe. Ovule clearing allows precise observation of whole tissues under Differential interference contrast (DIC) microscopy. The use of Methyl Salicylate as a clearing agent has proved to be particularly reliable for Solanaceous species.
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Visualization and Quantification of Actin Dynamics in Rice Protoplasts Authors:  Yurong Xie and Shanjin Huang, date: 11/05/2013, view: 4310, Q&A: 0
Direct visualization of the organization and dynamics of the actin cytoskeleton in rice cells is essential to understand its roles in regulating rice growth and development. Visualization of actin dynamics in protoplasts transformed with actin probe is a relatively quick and simple strategy, compared to the strategy of generating stable transgenic ...
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Stem Cell

In vivo BrdU Incorporation Assay for Murine Hematopioetic Stem Cells Authors:  Ningfei An and Yubin Kang, date: 11/05/2013, view: 8040, Q&A: 0
Bromodeoxyuridine (BrdU) is a thymidine analog that is incorporated into DNA during the S-phase of the cell cycle. As such, BrdU incorporation can be used to quantify the number of cells that are in S-phase in the time period during which BrdU is available. The following protocol describes an in vivo BrdU incorporation assay as a measure ...
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