Search
  • Volume 8, 2018
  • Volume 7, 2017
  • Volume 6, 2016
  • Volume 5, 2015
  • Volume 4, 2014
  • Volume 3, 2013
  • Volume 2, 2012
  • Volume 1, 2011

Developmental Biology

Terminal Deoxynucleotidyl Transferase Mediated Production of Labeled Probes for Single-molecule FISH or RNA Capture Authors:  Imre Gaspar, Frank Wippich and Anne Ephrussi, date: 03/05/2018, view: 1993, Q&A: 0
Arrays of short, singly-labeled ssDNA oligonucleotides enable in situ hybridization with single molecule sensitivity and efficient transcript specific RNA capture. Here, we describe a simple, enzymatic protocol that can be carried out using basic laboratory equipment to convert arrays of PCR oligos into smFISH and RAP probesets in a ...
More >>

Immunology

Quantification of Bacterial Attachment to Tissue Sections Authors:  Batya Isaacson, Tehila Hadad, Gilad Bachrach and Ofer Mandelboim, date: 03/05/2018, view: 1317, Q&A: 0
Here we describe a method to test bacterial adhesion to paraffin embedded tissue sections. This method allows examining binding of different bacterial strains, transfected with a fluorescent protein reporter plasmid to various tissues, to better understand different mechanisms such as colonization. This assay provides a more physiological context ...
More >>
Murine Pancreatic Islets Transplantation under the Kidney Capsule Authors:  Tatiana Jofra, Giuseppe Galvani, Fousteri Georgia, Gregori Silvia, Nicola Gagliani and Manuela Battaglia, date: 03/05/2018, view: 1501, Q&A: 0
Type 1 diabetes (T1D) is an autoimmune disease caused by the lack of insulin-producing pancreatic beta cells leading to systemic hyperglycemia. Pancreatic islet transplantation is a valid therapeutic approach to restore insulin loss and to promote adequate glycemic control. Pancreatic islet transplantation in mice is an optimal preclinical model ...
More >>
Mono Sodium Urate Crystal-induced Peritonitis for in vivo Assessment of Inflammasome Activation Authors:  Marianne R. Spalinger and Michael Scharl, date: 03/05/2018, view: 1604, Q&A: 0
Due to its particulate material, mono-sodium urate (MSU) crystals are potent activators of the NOD-like receptor NLRP3. Upon activation, NLRP3 induces the formation of inflammasome complexes, which lead to the production and release of mature IL-1β. Bioactive IL-1β is a potent activator of innate immune responses and promotes recruitment of ...
More >>
Intravenous Labeling and Analysis of the Content of Thymic Perivascular Spaces Authors:  Roland Ruscher and Kristin A. Hogquist, date: 03/05/2018, view: 1251, Q&A: 0
Following development in the thymus, T cells are thought to exit into the periphery predominantly through perivascular spaces (PVS). This exit route is used by conventional T cells, and likely also applies to unconventional T cell subsets, such as precursors of CD8αα and TCRγδ intraepithelial lymphocytes, regulatory T cells and natural killer T ...
More >>

Microbiology

Determination of Polyhydroxybutyrate (PHB) Content in Ralstonia eutropha Using Gas Chromatography and Nile Red Staining Authors:  Janina R. Juengert, Stephanie Bresan and Dieter Jendrossek, date: 03/05/2018, view: 1686, Q&A: 0
Ralstonia eutropha H16 produces and mobilizes (re-utilizes) intracellular polyhydroxybutyrate (PHB) granules during growth. This protocol describes the visualization of intracellular Nile red stained PHB granules and the quantification of PHB by gas chromatography. Our first method describes how to analyze PHB granules by fluorescence ...
More >>
Visualization of RNA 3’ ends in Escherichia coli Using 3’ RACE Combined with Primer Extension Authors:  Xun Wang, Heung Jin Jeon, Monford Paul Abishek N, Jin He and Heon M. Lim, date: 03/05/2018, view: 1378, Q&A: 0
In this assay, 3’ RACE (Rapid Amplification of cDNA 3’ Ends) followed by PE (primer extension), abbreviated as 3’ RACE-PE is used to identify the mRNA 3’ ends. The following protocol describes the amplification of the mRNA 3’ ends at the galactose operon in E. coli and the corresponding visualization of the PCR products through PE. In PE, ...
More >>
In vivo Analysis of Cyclic di-GMP Cyclase and Phosphodiesterase Activity in Escherichia coli Using a Vc2 Riboswitch-based Assay Authors:  Ying Liu, Hyunhee Kim and Ute Römling, date: 03/05/2018, view: 1350, Q&A: 0
Cyclic di-guanosine monophosphate (c-di-GMP) is a ubiquitous second messenger that regulates distinct aspects of bacterial physiology. It is synthesized by diguanylate cyclases (DGCs) and hydrolyzed by phosphodiesterases (PDEs). To date, the activities of DGC and PDE are commonly assessed by phenotypic assays, mass spectrometry analysis of ...
More >>

Neuroscience

Flight and Climbing Assay for Assessing Motor Functions in Drosophila Authors:  Steffy B Manjila and Gaiti Hasan, date: 03/05/2018, view: 1722, Q&A: 0
Motor control requires the central nervous system to integrate different sensory inputs and convey this information to the relevant central pattern generator for execution of motor function through motor neurons and muscles. Proper motor control is essential for any mobile organism to survive and interact with the external environment. For flying ...
More >>
Barnes Maze Procedure for Spatial Learning and Memory in Mice Author:  Matthew W. Pitts, date: 03/05/2018, view: 1843, Q&A: 0
The Barnes maze is a dry-land based rodent behavioral paradigm for assessing spatial learning and memory that was originally developed by its namesake, Carol Barnes. It represents a well-established alternative to the more popular Morris Water maze and offers the advantage of being free from the potentially confounding influence of swimming ...
More >>
A Fluorescent Dye Method Suitable for Visualization of One or More Rat Whiskers Authors:  Jacopo Rigosa, Alessandro Lucantonio, Giovanni Noselli, Arash Fassihi, Erik Zorzin, Fabrizio Manzino, Francesca Pulecchi and Mathew E Diamond, date: 03/05/2018, view: 995, Q&A: 0
Visualization and tracking of the facial whiskers is critical to many studies of rodent behavior. High-speed videography is the most robust methodology for characterizing whisker kinematics, but whisker visualization is challenging due to the low contrast of the whisker against its background. Recently, we showed that fluorescent dye(s) can be ...
More >>
Dual-sided Voltage-sensitive Dye Imaging of Leech Ganglia Authors:  Yusuke Tomina and Daniel A. Wagenaar, date: 03/05/2018, view: 1297, Q&A: 0
In this protocol, we introduce an effective method for voltage-sensitive dye (VSD) loading and imaging of leech ganglia as used in Tomina and Wagenaar (2017). Dissection and dye loading procedures are the most critical steps toward successful whole-ganglion VSD imaging. The former entails the removal of the sheath that covers neurons in the ...
More >>
Mouse Phrenic Nerve Hemidiaphragm Assay (MPN) Authors:  Giulia Zanetti, Samuele Negro, Marco Pirazzini and Paola Caccin, date: 03/05/2018, view: 1145, Q&A: 0
The neuromuscular junction (NMJ) is the specialized synapse by which peripheral motor neurons innervate muscle fibers and control skeletal muscle contraction. The NMJ is the target of several xenobiotics, including chemicals, plant, animal and bacterial toxins, as well as of autoantibodies raised against NMJ antigens. Depending on their ...
More >>
Construction and Cloning of Minigenes for in vivo Analysis of Potential Splice Mutations Authors:  Lisa Maria Riedmayr, Sybille Böhm, Stylianos Michalakis and Elvir Becirovic, date: 03/05/2018, view: 1504, Q&A: 0
Disease-associated mutations influencing mRNA splicing are referred to as splice mutations. The majority of splice mutations are found on exon-intron boundaries defining canonical donor and acceptor splice sites. However, mutations in the coding region (exonic mutations) can also affect mRNA splicing. Exact knowledge of the disease mechanism of ...
More >>

Plant Science

Large Scale Field Inoculation and Scoring of Maize Southern Leaf Blight and Other Maize Foliar Fungal Diseases Authors:  Shannon M. Sermons and Peter J. Balint-Kurti, date: 03/05/2018, view: 1282, Q&A: 0
Field-grown maize is inoculated with Cochliobolus heterostrophus, causal agent of southern leaf blight disease, by dropping sorghum grains infested with the fungus into the whorl of each maize plant at an early stage of growth. The initial lesions produce secondary inoculum that is dispersed by wind and rain, causing multiple cycles of ...
More >>
Histone Deubiquitination Assay in Nicotiana benthamiana Authors:  Shujing Liu and Lars Hennig, date: 03/05/2018, view: 771, Q&A: 0
Histone modifications are a group of post-translational modifications on histones which can alter chromatin structure and affect gene expression. Histone ubiquitination is a histone modification found in particular on histone H2A and H2B. Histone ubiquitination can be reversed by ubiquitin-specific proteases (UBP). Here, we describe an in vivo ...
More >>
Boron Uptake Assay in Xenopus laevis Oocytes Authors:  Sheliang Wang, Namiki Mitani-Ueno and Junpei Takano, date: 03/05/2018, view: 1327, Q&A: 0
Boron (B) is essential for plant growth and taken up by plant roots as boric acid. Under B limitation, B uptake and translocation in plants are dependent on the boric acid channels located in the plasma membrane. Xenopus leavis oocyte is a reliable heterologous expression system to characterize transport activities of boric acid channels ...
More >>
Quantification of Plant Cell Death by Electrolyte Leakage Assay Authors:  Noriyuki Hatsugai and Fumiaki Katagiri, date: 03/05/2018, view: 2742, Q&A: 0
We describe a protocol to measure the electrolyte leakage from plant tissues, resulting from loss of cell membrane integrity, which is a common definition of cell death. This simple protocol is designed to measure the electrolyte leakage from a tissue sample over a time course, so that the extent of cell death in the tissue can be monitored ...
More >>

Systems Biology

Coupling Exonuclease Digestion with Selective Chemical Labeling for Base-resolution Mapping of 5-Hydroxymethylcytosine in Genomic DNA Authors:  Aurélien A. Sérandour, Stéphane Avner and Gilles Salbert, date: 03/05/2018, view: 1401, Q&A: 0
This protocol is designed to obtain base-resolution information on the level of 5-hydroxymethylcytosine (5hmC) in CpGs without the need for bisulfite modification. It relies on (i) the capture of hydroxymethylated sequences by a procedure known as ‘selective chemical labeling’ (see Szulwach et al., 2012) and (ii) the digestion of the ...
More >>
Synthetic Genetic Interaction (CRISPR-SGI) Profiling in Caenorhabditis elegans Authors:  John A. Calarco and Adam D. Norris, date: 03/05/2018, view: 1521, Q&A: 0
Genetic interaction screens are a powerful methodology to establish novel roles for genes and elucidate functional connections between genes. Such studies have been performed to great effect in single-cell organisms such as yeast and E. coli (Schuldiner et al., 2005; Butland et al., 2008; Costanzo et al., 2010), ...
More >>