Isolation and Characterization of Dendritic Cells and…
Published 5/21/2012
Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction
Institute of Molecular Medicine and Genetics, Georgia Health Sciences University
To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.
Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes
STED Microscopy of Synaptic Function, European Neuroscience Institute Göttingen
FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.
Measuring Exocytosis in Neurons Using FM Labeling
Department of Molecular and Cell Biology, Harvard
The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.
Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties
Department of Physiology and Biophysics, Weill Cornell Medical College
We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.
Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development
1Department of Veterinary Science, University of Wisconsin, Madison, 2Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, 3Present address: Department of Pulmonary Medicine, University of Michigan, Ann Arbor, MI, 4Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI
A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.
In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons
Department of Biological Sciences, SUNY-University at Buffalo
This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.
Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes
Membrane Biology Group, Centre for integrative Physiology, University of Edinburgh
A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.
Measuring Peptide Translocation into Large Unilamellar Vesicles
1Department of Chemistry, Wellesley College,
This protocol details a method for the quantitative measure of peptide translocation into large unilamellar lipid vesicles. This method also provides information about the rate of membrane translocation and can be used to identify peptides that efficiently and spontaneously cross lipid bilayers.
Physiological Recordings of High and Low Output NMJs on the Crayfish Leg Extensor Muscle
Department of Biology, University of Kentucky
This article demonstrates how to conduct electrophysiological recordings of synaptic responses on the extensor muscle in the walking leg of a crayfish and how the nerve terminals are visualized to show the gross morphological differences of high- and low-output nerve terminals.
Culture of Mouse Neural Stem Cell Precursors
1Department of Developmental and Cell Biology, University of California, Irvine (UCI), 2Department of Pathology, University of California, Irvine (UCI), 3Department of Physiology and Biophysics, University of California, Irvine (UCI)
This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.
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