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Biochemistry

Immunolabeling of Proteins in situ in Escherichia coli K12 Strains Authors:  Nienke Buddelmeijer, Mirjam Aarsman and Tanneke den Blaauwen, date: 08/05/2013, view: 4383, Q&A: 0
This protocol was developed to label proteins in bacterial cells with antibodies conjugated to a fluorophore for fluorescence microscopy imaging. The procedure is optimized to minimize morphological changes and also to minimize the amount of antibodies needed for the staining. The protocol can also be used with primary antibodies conjugated to a ...
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Cancer Biology

Generation of Mouse Lung Epithelial Cells Authors:  Andrea L. Kasinski and Frank J. Slack, date: 08/05/2013, view: 5494, Q&A: 0
Although in vivo models are excellent for assessing various facets of whole organism physiology, pathology, and overall response to treatments, evaluating basic cellular functions, and molecular events in mammalian model systems is challenging. It is therefore advantageous to perform these studies in a refined and less costly setting. One ...
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Retrovirus Mediated Malignant Transformation of Mouse Embryonic Fibroblasts Authors:  Huei San Leong and Marnie Blewitt, date: 08/05/2013, view: 4609, Q&A: 2
Cellular transformation is a widely used method to artificially induce cells to form tumours in vivo. Here, we describe the methodology for malignant transformation of mouse embryonic fibroblasts (MEFs) for transplantation into immunodeficient nude mice, as used in Leong et al. (2013). The two-step process involves: 1) ...
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Cell Biology

Extravillous Trophoblast Migration and Invasion Assay Authors:  Magdalena Angelova, Heather L. Machado, Kenneth F. Swan, Cindy Morris and Deborah E. Sullivan, date: 08/05/2013, view: 4473, Q&A: 0
Extravillous trophoblast (EVT) migration and invasion through the decidualized endometrium is essential to successful placentation. SGHPL-4 cells, an EVT cell line derived from first trimester placenta, is a widely used model of cytotrophoblast differentiation into an invasive phenotype. Here we describe a quantitative cell migration assay that ...
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Assay of Blood Brain Barrier and Placental Barrier Permeability Authors:  Saurabh Kumar Agnihotri, Poonam Singh, Balawant Kumar, Pankaj Singh, Swatantra Kumar Jain, Mahesh Chandra Tewari, Sadan Kumar, Monika Sachdev and Raj Kamal Tripathi, date: 08/05/2013, view: 5008, Q&A: 0
Evans blue dye solution was used to observe the effect of a viral protein on two pre-defined barriers of the body i.e. the blood brain barrier (BBB) and the placental barrier (PB). This dye has strong affinity for serum albumin and does not cross these barriers under natural conditions. As all the dye gets bound to albumin, all the neural ...
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Epidermal Growth Factor (EGF) Receptor Endocytosis Assay in A549 Cells Authors:  Sabrina Rizzolio and Luca Tamagnone, date: 08/05/2013, view: 8431, Q&A: 0
The following endocytosis assay has been optimized to assess EGF-stimulated EGFR endocytosis; but could be modified to assess other ligand-stimulated endocytosis of plasma membrane receptors (for which fluorochrome-conjugated ligands are available to track their receptor internalization). In brief, cells are treated with fluorescent EGF at 4 °C to ...
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Immunology

Whole Spleen Flow Cytometry Assay Authors:  Cathy S. Yam and Adeline M. Hajjar, date: 08/05/2013, view: 12711, Q&A: 0
In the Whole Spleen Flow Cytometry Assay, we used splenocytes directly ex vivo for stimulation with a variety of TLR ligands. The splenocytes were stimulated for a total of 4 hours, then stained for intracellular cytokines. We then examined cytokine production via flow cytometry. This allowed us to compare the responses of minimally ...
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Transfection of Human Naive CD4+ T Cells with PHA Activation and Neon Electroporation Authors:  Amy Palin and David B. Lewis, date: 08/05/2013, view: 7921, Q&A: 0
Transfection of primary T cells can be challenging. This protocol describes a method to transfect primary human naive CD4+ T cells with an AP-1 luciferase reporter using low-level activation by phytohemagglutinin (PHA) and electroporation, as published (Palin et al., 2013). This technique is a modification of one previously ...
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Pulmonary Myeloperoxidase Activity Author:  Tammy Regena Ozment, date: 08/05/2013, view: 4338, Q&A: 0
Neutrophils are considered one of the first responders of the innate immune response. Their primary activities are to migrate to sites of infection by chemotaxis and trans-migration across the endothelium (Gaines et al., 2005). Once at the site of infection, they phagocytize microbes and kill them. Critical to the neutrophil’s ability to ...
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Microbiology

EMSA Analysis of DNA Binding By Rgg Proteins Authors:  Breah LaSarre and Michael J. Federle, date: 08/05/2013, view: 6423, Q&A: 0
In bacteria, interaction of various proteins with DNA is essential for the regulation of specific target gene expression. Electrophoretic mobility shift assay (EMSA) is an in vitro approach allowing for the visualization of these protein-DNA interactions. Rgg proteins comprise a family of transcriptional regulators widespread among the ...
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CAMP-Membrane Interactions Using Fluorescence Spectroscopy Authors:  Ron Saar-Dover and Yechiel Shai, date: 08/05/2013, view: 3875, Q&A: 0
The molecular mechanism by which peptide antibiotics (also referred as cationic antimicrobial peptides-CAMPs) penetrate through the bacterial wall barrier, interact with, and disrupt their membrane is complex. It depends mainly on the peptide properties (structure, length, charge and hydrophobicity), on the characteristics of the cell wall matrix ...
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Molecular Biology

Total RNA Isolation after Laser-capture Microdissection of Human Cervical Squamous Epithelial Cells from Fresh Frozen Tissue Authors:  Saskia M Wilting and Renske DM Steenbergen, date: 08/05/2013, view: 5395, Q&A: 0
As most tissue specimens contain a mixture of different cell types including epithelial cells, stromal cells and immune cells, selection of the cells of interest is of utmost importance for the accurate determination of gene/microRNA expression. Laser capture microdissection enables the researcher to obtain homogeneous ultrapure cell selections ...
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Neuroscience

Subcellular Fractionation of Mouse Brain Homogenates Authors:  Ditte Olsen and Camilla Gustafsen, date: 08/05/2013, view: 6347, Q&A: 0
This subcellular fractionation protocol is used for separation of cellular organelles based on their density. We have designed and optimized the protocol for separation of subcellular compartments of brain homogenates with focus on the localization and trafficking of transmembrane proteins, but we have also successfully used this protocol for ...
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In vivo Neurogenesis Authors:  Désirée R. M. Seib and Ana Martin-Villalba, date: 08/05/2013, view: 5750, Q&A: 0
This protocol shows how to characterize the dynamics of hippocampal neurogenesis in the adult mouse by describing preparation of brain tissue, immunofluorescence of brain sections and confocal stereotactic cell counting.
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Neuronal Morphology Analysis Authors:  Désirée R. M. Seib and Ana Martin-Villalba, date: 08/05/2013, view: 5343, Q&A: 0
This protocol describes how to visualize neuronal morphology and how to determine neuronal complexity of immature and mature hippocampal neurons in the mouse in vivo including tissue preparation, staining of brain sections and confocal cell analysis.
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Isolation of Growth Cones from Mouse Brain Authors:  Iryna Leshchyns’ka and Vladimir Sytnyk, date: 08/05/2013, view: 4808, Q&A: 0
Growth cones are motile structures at the tips of growing neurites, which play an essential role in regulation of growth and navigation of growing axons and dendrites of neurons in the developing nervous system. This protocol describes isolation of growth cones from the brain tissue from young mice. Growth cones isolated using this protocol have ...
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Plant Science

Maize Kernels – Fixation in FAA, Embedding, Sectioning and Feulgen Staining Author:  Aleš Kladnik, date: 08/05/2013, view: 7023, Q&A: 0
The protocol describes preparation of young developing maize kernels for microscopical analysis of nuclei in tissue sections. The fixative FAA (formaldehyde, acetic acid and ethanol) is suitable for preservation of nuclear morphology and allows for quantitative staining of DNA with Schiff reagent in Feulgen staining. The fixation and embedding ...
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Determination of Ferric Chelate Reductase Activity in the Arabidopsis thaliana Root Authors:  Emre Aksoy and Hisashi Koiwa, date: 08/05/2013, view: 6369, Q&A: 5
Plants have developed two distinct mechanisms, i.e., strategy I (reduction strategy) and II (chelation strategy), to mobilize insoluble Fe(III) in the rhizosphere and transport it through the plasma membrane. Arabidopsis thaliana and other dicots rely on strategy I. In this strategy, the rhizosphere is first acidified by a ...
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Heat Shock Treatment of Chlamydomonas reinhardtii and Chlorella Cells Authors:  Stephanie Chankova, Zhana Mitrovska and Nadezhda Yurina, date: 08/05/2013, view: 4125, Q&A: 0
The protocol is very reliable and simple for inducing heat shock in unicellular green algae cells. The main purpose was to compare cellular response of three Chlorella species, isolated from different habitats: Chlorella vulgaris 8/1- thermophilic, Chlorella kesslery- mesophilic and C. vulgaris- extremophilic. ...
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Western Blot Analysis of Chloroplast HSP70B in Chlorella Species Authors:  Stephka Chankova, Zhana Mitrovska and Nadezhda Yurina, date: 08/05/2013, view: 4377, Q&A: 0
Western blotting allows for the specific detection of proteins by an antibody of interest. This protocol utilizes isolation of total proteins protocol for Chlorella vulgaris prior to gel electrophoresis. After electrophoresis, the selected antibodies are used to detect and quantify levels of chloroplast HSP70B.
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