1Department of Chemistry, University of Wisconsin- Madison, 2School of Pharmacy, University of Wisconsin- Madison
Mass spectrometric imaging (MSI) is a powerful tool that can be used to discover and identify various chemical species in intact tissues, preserving the compounds in their native environments, which can provide new insights into biological processes. Herein a MSI method developed for the analysis of small molecules is described.
Published March 5, 2014. Keywords: Basic Protocol, Mass Spectrometric Imaging, Imaging Mass Spectrometry, MALDI, TOF/TOF, Medicago truncatula, Metabolite, Small Molecule, Sublimation, Automatic Sprayer
1Institut Curie, Centre Universitaire, 2Université Paris Sud, Centre Universitaire, 3CNRS UMR 3347, Centre Universitaire, 4INSERM U1021, Centre Universitaire
This protocol describes how to dissect premigratory cranial neural crest (NC) from Xenopus laevis neurulas. These explants can be plated on fibronectin and cultured in vitro, or grafted back into host embryos. This technique allows studying the mechanisms of NC epithelium-to-mesenchyme transition, migration, and differentiation.
Published March 4, 2014. Keywords: Developmental Biology, Neural crest, Xenopus laevis, embryo, dissection, graft, fibronectin
1Neuroscience Division of the Biomedical & Health Sciences Institute, University of Georgia, 2Department of Cellular Biology, University of Georgia
Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr exhibit morphological development comparable to embryos developing in utero.
Published March 1, 2014. Keywords: Developmental Biology, mouse embryo, mid-gestation, serum-free, defined media, roller culture, organogenesis, development
1Animal Bioscience and Biotechnology Laboratory, United States Department of Agriculture, 2Department of Animal and Avian Sciences, University of Maryland
Utero-tubal embryo transfer uses the utero-tubal junction as a barrier to prevent the embryo outflow that may occur when performing uterine transfer. Vasectomized males are required to obtain pseudopregnant recipients for embryo transfer. Both techniques are discussed.
Published February 28, 2014. Keywords: Basic Protocols, blastocyst, chimera, lentivirus, uterine transfer, oviductal transfer, utero-tubal transfer
1Huisken Lab, Max Planck Institute of Molecular Cell Biology and Genetics
The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.
Published February 27, 2014. Keywords: Developmental Biology, zebrafish, Danio rerio, light sheet microscopy, Selective Plane Illumination Microscopy, sample mounting, time lapse microscopy, long-term imaging
1Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center, 2Department of Physiology and Cell Biology, Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center, 3Department of Molecular Biophysics and Physiology, Rush University Medical Center, 4Department of Internal Medicine, Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center
Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.
Published February 24, 2014. Keywords: Physiology, flexor digitorm brevis (FDB), sarcoplasmic reticulum, SR Ca2+ release, calcium signaling, ryanodine receptor, confocal imaging, muscle physiology
1School of Life Sciences, University of Warwick, 2The Sainsbury Laboratory, Norwich Research Park, 3Research School of Biology, The Australian National University
We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.
Published February 22, 2014. Keywords: Plant Biology, plant-microbe interactions, protein complex purification, mass spectrometry, protein phosphorylation, Prf, Pto, AvrPto, AvrPtoB
1Fondazione Filarete, 2Department of Biotechnology and Translational Medicine, University of Milan, 3Neuroscience Institute, National Research Council (CNR), 4Department of Health Science, "Magna Graecia" University of Catanzaro
We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.
Published February 18, 2014. Keywords: Microbiology, Endoplasmic reticulum (ER), fluorescent proteins (FPs), confocal microscopy, fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), ultrastructure, transmission electron microscopy (TEM)
1Laboratoire de Cristallographie & RMN Biologiques, CNRS & Université Paris Descartes
We present a protocol for the in vitro investigation of efflux pumps from Pseudomonas aeruginosa. This protocol allows for the generation of a robust, reversible, and tunable proton gradient within the liposome membrane and hence should be adaptable to any membrane protein energized by the protomotive force.
Published February 17, 2014. Keywords: Biochemistry, membrane protein, transport, antibiotic resistance, liposomes, proton gradient, bacteriorhodopsin
1Department of Horticulture, University of Kentucky
Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.
Published February 16, 2014. Keywords: Biochemistry, Affinity selection, Phage display, protein-protein interaction