Name: quantifying synapses: an immunocytochemistry-based assay to quantify synapse number
Uploaded: 2010-11-16T17:30:00
Description: Watch this Scientific Journal Video about Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number at JoVE.com

Puncta Analyzer plug-in voor Image J werd door Barry Wark (huidig ​​adres: Physion Consulting) geschreven in het lab van Ben A. Barres (Stanford University). Financiering; <ul><li> Alfred P. Sloan Foundation </li><li> De Esther A. en Joseph Klingenstein Fund, Inc </li><li> Brede Biomedical Research Foundation </li><li> Remedie voor de ziekte van Huntington s Initiatief </li></ul>

Oplossingen voor te bereiden: <ol><li> Antilichaam Buffer: <ul><li> 150 mM NaCl </li><li> 50 mM Tris-Base (Fisher, Cat No:. BP152-5, 50 mm) - 1,21 g </li><li> 1% BSA (Sigma, Cat No:. A2153, 1%) - 2,0 g </li><li> 100 mM L-lysine (Sigma, Cat No:. L-1137, 100 mm) - 3,65 g </li><li> Pas de pH tot 7,4 </li><li> 0,04% Azide </li><li> Stel het volume tot 200 ml met gedestilleerd H 2 O. </li><li> Filtreer door 0.22μm filter (Millipore, Cat No:. SCGPU02RE). </li></ul></li><li> PFA verdunningsmiddel: <ul>...

<ol>
	<li>Barres, B. A., Silverstein, B. E., Corey, D. P., Chun, L. L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological,+morphological,+and+electrophysiological+variation+among+retinal+ganglion+cells+purified+by+panning.">Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning.</a> Neuron. 1, 791-803 (1988).</li><li>Meyer-Franke, A., Kaplan, M. R., Pfrieger, F. W., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+signaling+interactions+that+promote+the+survival+and+growth+of+developing+retinal+ganglion+cells+in+culture.">Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture.</a> Neuron. 15, 805-819 (1995).</li><li>Eroglu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gabapentin+receptor+alpha2delta-1+is+a+neuronal+thrombospondin+receptor+responsible+for+excitatory+CNS+synaptogenesis.">Gabapentin receptor alpha2delta-1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis.</a> Cell. 139, 380-392 (2009).</li><li>Pfrieger, F. W., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+efficacy+enhanced+by+glial+cells+in+vitro.">Synaptic efficacy enhanced by glial cells in vitro.</a> Science. 277, 1684-1687 .</li><li>Ullian, E. M., Sapperstein, S. K., Christopherson, K. S., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+synapse+number+by+glia.">Control of synapse number by glia.</a> Science. 291, 657-661 (2001).</li><li>Christopherson, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombospondins+are+astrocyte-secreted+proteins+that+promote+CNS+synaptogenesis.">Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis.</a> Cell. 120, 421-433 (2005).</li><li>Godement, P., Salaun, J., Imbert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prenatal+and+postnatal+development+of+retinocollicular+projections+in+the+mouse.">Prenatal and postnatal development of retinocollicular projections in the mouse.</a> J. Comp. Neurol. 230, 552-575 (1985).</li><li>Schmidt, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+retinotopic+connections:+selective+stabilization+by+an+activity-dependent+mechanism.">Formation of retinotopic connections: selective stabilization by an activity-dependent mechanism.</a> Cell. Mol. Neurobiol. 5, 65-84 (1985).</li><li>Sachs, G. M., Jacobson, M., Caviness, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+changes+in+arborization+patterns+of+murine+retinocollicular+axons.">Postnatal changes in arborization patterns of murine retinocollicular axons.</a> J. Comp. Neurol. 246, 395-408 (1986).</li><li>Elmariah, S. B., Oh, E. J., Hughes, E. G., Balice-Gordon, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+regulate+inhibitory+synapse+formation+via+Trk-mediated+modulation+of+postsynaptic+GABAA+receptors.">Astrocytes regulate inhibitory synapse formation via Trk-mediated modulation of postsynaptic GABAA receptors.</a> J. Neurosci. 25, 3638-3650 (2005).</li><li>Hughes, E. G., Elmariah, S. B., Balice-Gordon, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+secreted+proteins+selectively+increase+hippocampal+GABAergic+length,+branching+and+synaptogenesis.">Astrocyte secreted proteins selectively increase hippocampal GABAergic length, branching and synaptogenesis.</a> Moll. Cell. Neurosci. 43, 136-145 (2010).</li></ol>

De synaps assay hierboven beschreven is gevestigd in de context van onze experimentele doelen, waarin we vooral focussen op prikkelende projecties van RGC, hetzij in gezuiverde cultuur of in de hersenen sectie. We hebben een referentie tabel met antilichamen die goed werken voor etikettering excitatoire synapsen (tabel 1). Dit synaps assay kan worden aangepast aan het aantal synaps van een neuronale populatie of een ander subtype synaptic waarvoor er een selectieve pre-en postsynaptische mar...

<table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td> Antigeen </td><td> Soorten </td><td> Monoklonale / Polyklonale </td><td> Verkoper </td><td> Catalogusnummer </td><td> Verdunning </td><td> Werken in Cultuur </td><td> Werken in de artikelen </td></tr><tr><td rowspan="10"> Presynaptische </td><td> Synapsin </td><td> Konijn </td><td> Polyklonale </td><td> Synaptic Systems </td><td> 106004 </td><td> 1:750 </td><td> Y </td><td> ND </td></tr><tr><td> Synapsin </td><td> Muis </td><td> Monoklonale </td><td> Synaptic Systems </td><td> 106001 </td><td> 1:500 </td><td> Y </td><td> Y </td></tr><tr><td> Fagot </td><td> Muis </td><td> Monoklonale </td><td> Assay Designs </td><td> VAM-PS003F </td><td> 1:500 </td><td> Y </td><td> Y </td></tr><tr><td> Fagot </td><td> Cavia </td><td> Polyklonale </td><td> Synaptic Systems </td><td> 141004 </td><td> 1:1000 </td><td> Y </td><td> ND </td></tr><tr><td> Synaptotagmin een </td><td> Konijn </td><td> Polyklonale </td><td> Synaptic Systems </td><td> 105002 </td><td> 1:750 </td><td> Y </td><td> N </td></tr><tr><td> Synaptobrevin 2 (C1.69.1) </td><td> Muis </td><td> Monoklonale </td><td> Synaptic Systems </td><td> 104211 </td><td> 1:500 </td><td> Y </td><td> Y </td></tr><tr><td> Synaptofysine (C1.7.2) </td><td> Muis </td><td> Monoklonale </td><td> Synaptic Systems </td><td> 101011 </td><td> 1:500 </td><td> Y </td><td> Y </td></tr><tr><td> VGlut1 </td><td> Muis </td><td> Monoklonale </td><td> Millipore </td><td> MAB5502 </td><td> 1:2500 </td><td> N </td><td> Y </td></tr><tr><td> VGlut1 </td><td> Cavia </td><td> Polyklonale </td><td> Millipore </td><td> AB5905 </td><td> 1:2500 </td><td> N </td><td> Y </td></tr><tr><td> VGlut2 </td><td> Cavia </td><td> Polyklonale </td><td> Millipore </td><td> AB2251 </td><td> 1:2500 </td><td> N </td><td> Y </td></tr><tr><td rowspan="6"> Postsynaptische </td><td> PSD-95 (6G6-1C9 kloon) </td><td> Muis </td><td> Monoklonale </td><td> Affiniteit Bio Reagentia </td><td> MA1-045 </td><td> 1:750 </td><td> Y </td><td> N </td></tr><tr><td> PSD-95 </td><td> Konijn </td><td> Polyklonale </td><td> Zymed </td><td> 51-6900 </td><td> 1:500 </td><td> N </td><td> Y </td></tr><tr><td> Homerus </td><td> Muis </td><td> Monoklonale </td><td> Synaptic Systems </td><td> 160011 </td><td> 1:500 </td><td> Y </td><td> ND </td></tr><tr><td> Homerus </td><td> Rat </td><td> Polyklonale </td><td> Millipore </td><td> AB5875 </td><td> 1:500 </td><td> Y </td><td> Y </td></tr><tr><td> Gephyrin </td><td> Konijn </td><td> Polyklonale </td><td> Synaptic Systems </td><td> 147003 </td><td> 1:500 </td><td> Y </td><td> Y </td></tr><tr><td> Gephyrin </td><td> Muis </td><td> Monoklonale </td><td> Synaptic Systems </td><td> 147 </td><td> 1:200 </td><td> Y </td><td> Y </td></tr></tbody></table> Tabel 1: Geeft een overzicht van voorbeelden van goede pre-en postsynaptische markeringen die we met succes hebben gebruikt in onze synaps test. Wees ervan bewust dat dit geen uitputtende lijst van alle beschikbare markers. Y = ja, N = Nee, ND = niet bepaald. <table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td> Reagens </td><td> Vennootschap </td><td> Cat. Nee. </td></tr><tr><td> PBS </td><td> Invitrogen </td><td> 20012-027 </td></tr><tr><td> poly-d-lysine </td><td> Sigma </td><td> P6407 </td></tr><tr><td> Laminine </td><td> Cultrex </td><td> 3400-010-01 </td></tr><tr><td> Triton X-100 </td><td> Roche Diagnostics Gmbh </td><td> 9002-93-1 </td></tr><tr><td> Normaal Geit Serum </td><td> Gibco </td><td> 16210 </td></tr><tr><td> VectaShield met DAPI </td><td> Vector Laboratories </td><td> H-1200 </td></tr><tr><td> Oktober </td><td> Tissue-Tek </td><td> 4583 </td></tr><tr><td> Tris-Base (50 mm) </td><td> Visser </td><td> BP152-5 </td></tr><tr><td> Bovine Serum Albumine </td><td> Sigma </td><td> A2153 </td></tr><tr><td> L-lysine </td><td> Sigma </td><td> L-1137 </td></tr><tr><td> 16% PFA-oplossing </td><td> Electron Microscopy Sciences </td><td> 15711 </td></tr><tr><td> Granular PFA </td><td> Electron Microscopy Sciences </td><td> 19210 </td></tr><tr><td> 24-well plaat cultuur </td><td> Valk </td><td> 35-3047 </td></tr><tr><td> Geit anti-muis Alexa geconjugeerde antilichamen </td><td> Invitrogen </td><td> --- </td></tr><tr><td> Leveren </td><td> Vennootschap </td><td> Cat. Nee. </td></tr><tr><td> 12mm, nr. 0 dekglaasjes </td><td> Karl Hecht Gmbh </td><td> 1105209 </td></tr><tr><td> Nee 1,5 glazen dekglaasje (voor plakjes) </td><td> VWR Scientific </td><td> 48393241 </td></tr><tr><td> Objectglaasjes </td><td> VWR Scientific </td><td> 48311-703 </td></tr></tbody></table>

quantifying synapses: an immunocytochemistry-based assay to quantify synapse number

Een van de belangrijkste doelstellingen in de neurowetenschappen is het begrijpen van de moleculaire signalen dat de vroege stadia van synapsvorming instrueren. Als zodanig is noodzakelijk geworden om objectieve benaderingen te ontwikkelen voor veranderingen in de synaptische verbindingen te kwantificeren. Vanaf monster fixatie, dit protocol beschreven hoe u synaps aantal kwantificeren, zowel in gedissocieerde neuronale cultuur en in de hersenen secties met behulp van immunocytochemie. Met behulp van vak-specifieke antilichamen, we label presynaptische terminals alsmede ten aanzien sites van postsynaptische specialisatie. We definiëren synapsen als punten van colocalization tussen de signalen die door deze markers. Het aantal van deze colocalizations wordt gekwantificeerd met behulp van een plug in Puncta Analyzer (geschreven door Bary Wark, beschikbaar op aanvraag, c.eroglu @ cellbio.duke.edu) onder de ImageJ analyse software platform. De synaps assay beschreven in dit protocol kan worden toegepast op alle neurale weefsel of de bereiding waarvoor u selectief pre-en postsynaptische markers. Dit synaps test is een waardevol instrument dat op grote schaal kan worden gebruikt in de studie van synaptische ontwikkeling.

Watch this Scientific Journal Video about Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number at JoVE.com

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

Dit protocol beschrijft hoe u synaps nummer zowel in gedissocieerde neuronale cultuur en in de hersenen secties met behulp van immunocytochemie kwantificeren. Met behulp van vak-specifieke antilichamen, we label presynaptische terminals alsmede ten aanzien sites van postsynaptische specialisatie. We definiëren synapsen als punten van colocalization tussen de signalen die door deze markers.

Het kwantificeren van Synapsen: een immunocytochemie-gebaseerde test te Synapse Number Kwantificeer

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

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