Name: high-resolution quantitative immunogold analysis of membrane receptors at retinal ribbon synapses
Uploaded: 2016-02-18T14:00:00.000+00:00
Duration: 9 min 24 s
Description: Watch this Scientific Journal Video about High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses at JoVE.com

Diese Arbeit wurde durch die Interne Programme des National Institute of Neurological Disorders and Stroke (NINDS) und National Institute on Taubheit und andere Communication Disorders (NIDCD), von den National Institutes of Health (NIH) unterstützt. Wir danken dem NINDS EM Anlage und die NIDCD Advanced Imaging-Kern (Code # ZIC DC 000081-03) um Hilfe.

Pflege und Betreuung von Tieren waren nach NIH Animal Care und Use Committee Guidelines. 12-Stunden-Licht: postnatalen Tag (P) 15-21 Sprague-Dawley-Ratten injiziert mit 1-1,2% CTB bilateral durch den Colliculus superior, wurden auf einem 12 gehalten Dunkel-Zyklus. 1. Netzhautgewebe Fixation <ol><li> Setzen Sie die folgenden Materialien und Werkzeuge: einem Binokular, 2 Zangen mit sehr feinen Spitzen, Schere, Zellulosefilterpapier, Plastikpipette und einem Objektträger. </li><li> Anesthetize die Ratte in einer geschlossenen Kammer mit 2,0 ml Halothan (ein Inhalationsanästhesie). Bestimmen ausreichende Betäubung mit diesen Methoden: Mangel Zurücknahme der Hinterpfote nach Zeh Prise, oder das Fehlen von Blinkreflex. Dann enthaupten sofort mit einer Guillotine. Entfernen Sie die Augen mit einem Paar von Iris Schere und in eine Glasschale mit 4% Paraformaldehyd in 0,1 M Phosphatpuffer (PB) bei pH 7,4. </li><li> das Binokular Entfernen Sie mit den Maisea durch die Vorderseite des Augapfels abzuschneiden. Entfernen Sie die Linse und Glas von der inneren Netzhautoberfläche mit einer Pinzette. </li><li> Ziehen Sie die Lederhaut mit den beiden Zangen, bis die Netzhaut von der Augenmuschel isoliert ist. </li><li> Schneiden Sie die Netzhaut sofort in 100 bis 200 &amp; mgr; m-dicke Streifen mit einer Rasierklinge, und je nach pH-Shift-Fixierung. </li><li> Fix Retina Streifen in 4% Paraformaldehyd in 0,1 M PB bei pH 6,0 für 20 - 30 min und dann in 4% Paraformaldehyd und 0,01% Glutaraldehyd bei pH 10,5 für 10 - 20 min bei RT. </li><li> Nach mehrmaligem Waschen in PB mit 0,15 mM CaCl 2 (pH 7,4 bei 4 ° C), cryoprotect die Netzhautstreifen mit Glycerin (60 min jeweils in 10%, 20%, 30%, dann O / N in 30%) in 0,1 M PB vor Substitution einzufrieren. </li></ol> 2. Gefriersubstitution HINWEIS: Diese gefrierSubstitutionsMethode aus einer früheren veröffentlichten Protokoll 19,20 modifiziert wird. Auch ist es von entscheidender Bedeutung, dass die Instrumente sehr kalt sind (Handschuhe); Otherwise, kann das Gewebe teilweise aufzutauen, als mit den Instrumenten berührt. Alle diese Schritte werden in der AFS Kammer und der sie durchgeführt werden niemals über den Rand der Kammer bewegen kann. In ähnlicher Weise eine ausreichende Kühlung aller im AFS verwendeten Chemikalien notwendig. <ol><li> Stech einfrieren die Netzhautstreifen in flüssigem Propan bei -184 ° C in einem EM Kryokonservierung Instrument (CPC). Mit einem feinen Pinsel, legen Sie die Proben auf kleine Stücke von doppelseitigem Klebeband auf die Metallstümpfe befestigt, die auf dem Ende der Eintauchstangen gehen (Docht aus zusätzlichen Puffer mit dem Pinsel). </li><li> Tauchen Sie die Stangen in die flüssige Propan und halten es für ca. 5 sec, und dann übertragen auf die automatische gefrier Substitution Instrument (AFS) ein kleines Transportkammer verwenden, die mit flüssigem Stickstoff ( &quot;LN 2 gekühlt Kryo Transferbehälter &#39;) gefüllt ist. </li><li> die gefrorene Probe und Instrumente (Zange und Skalpell) in den AFS, kühlen die Instrumente in der Kammer nach der Platzierung für seVeral Minuten, bevor Sie das Gewebe zu berühren, oder sie kühlen, indem die Spitzen der Instrumente für ein paar Sekunden in die kleine Transportkammer platzieren (gefüllt mit flüssigem Stickstoff ( &quot;LN 2 gekühlt Kryo Transferbehälter &#39;)). </li><li> Einmal in der AFS, entfernen Sie die Probe und das Klebeband von der Stummel einer feinen Skalpell. Auch halten die Stickstoffgasflusssteuerung, TF (TF wird als &quot;Regler-Steuerung für LN2 Verdampfer&quot; bezeichnet) geöffnet während dieser Vorgänge. </li><li> Vor dem gefrorenen Gewebe in die Flach Einbettung Halter zu platzieren (dh vor den AFS-Einrichtung), Schnitt einen dünnen Kreis aus einem klaren Kunststofffolie und legen Sie sie in den unteren Teil der Halterung, um den Boden jeder Vertiefung zu füllen. Auf diese Weise können relativ leichteres Entfernen der polymerisierten Blöcken Probe, wenn Sie fertig. HINWEIS: Früher haben wir genutzt, ein alternatives Verfahren zu den flachen Einbettung Halter und Doppel Reichert + Gelatinekapseln unter Verwendung (siehe Petralia und Wenthold, 1999) &lt;sup&gt; 20. </li><li> Verwenden Sie die folgende automatische Sequenz in der AFS (unter Verwendung Instrument Terminologie): T1 = -90 ° C für 32 h, S1 = Zunahme Temperatur von 4 ° C / h (11,3 h), T2 = 45 ° C für 50 h, S2 = Temperaturanstieg von 5 ° C / h (9 hr), T3 = 0 ° C für 40 Stunden (gesamt = 142,3 h). 4 Stunden Proben in der AFS zu platzieren (bei -90 ° C), bevor die Zeitabfolge beginnen - Es kann 2 nehmen. </li><li> Tauchen die Gefrierschnitte in 1,5% Uranylacetat in Methanol bei -90 ° C für&gt; 32 Stunden in dem AFS. Legen Sie zwei Stücke von Gewebe (in der Regel) in jedem der sieben Brunnen im Flach Einbettung Aluminiumhalter (drei passen in die AFS). <ol><li> Platzieren der Gewebe + Band in das Uranylacetat / Methanol in den Vertiefungen und entfernen das Band später, unmittelbar vor dem Beginn Einbettungsmedium (zB Lowicryl HM20) Infiltration, wenn es zu schwierig ist, das Gewebe von dem Band zu entfernen. </li></ol></li><li> Dann wird die Temperatur schrittweise erhöhen bis -45 ° C (+ 4 ° C pro Stunde; in der automatischen Sequenz). </li><li> Waschen Sie die Proben dreimal in vorgekühlte Methanol, um einen spitzen Plastikpipette mit der alten Lösung von jedem Flach Einbettung Halter, und dann mit einem anderen Standard-Kunststoff-Pipette zu entfernen Sie die vorgekühlt frischem Methanol hinzuzufügen. </li><li> Dann mit der gleichen Methode, infiltrieren die Proben schrittweise mit niedriger Temperatur Einbettung Harze wie Einbettungsmedium (HM20 / Methanol im Verhältnis 1: 1 und 2: 1, jeweils für 2 Stunden, gefolgt von Reinharz für 2 Stunden und dann die Harze ändern und halten O / N). </li><li> Ändern Sie den Harz am nächsten Tag wieder, das Niveau des Harzes Einstellung nur bis zur Oberkante der Brunnen zu erreichen. </li><li> Polymerisation der Proben (-45 ° C bis 0 ° C in automatischer Folge; + 5 ° C pro h) mit UV-Licht für 40 Stunden. </li><li> Dann entfernen Sie die Proben aus der AFS. Typischerweise zeigen Probenblöcke noch etwas rosa Haut und in Farbe. Legen Sie die Proben der Nähe des Leuchtstofflampe in der chemischen Abzugshaube, bei RT O / N oder mehr, bis sie AppOhr ganz klar. </li></ol> 3. Postembedding EM Immungold Immunzytochemie HINWEIS: Postembedding Immunzytochemie wird wie beschrieben durchgeführt 23,24,25. <ol><li> Cut 1 &amp; mgr; m Abschnitte mit Ultramikrotoms, Fleck Abschnitte mit 1% Toluidin-Blau, und untersuchen sie für Abschnitt Orientierung; Ausrichten der Gewebeblock, um die optimale Querebene Schnitt erreichen. </li><li> Cut 70 nm dicke Ultradünnschnitten mit Ultramikrotoms und sammeln sie auf Formvar-Kohlenstoff beschichteten Nickel-Spiel Gitter. </li><li> Wash Gitter einmal in destilliertem H 2 O durch eine Tris-gepufferte Kochsalzlösung, gefolgt dreimal (TBS, 0,05 M Tris-Puffer, 0,7% NaCl, pH 7,6) waschen. </li><li> Inkubieren Gitter in 20 ul Tropfen 5% BSA in TBS für 30 min und dann in 20 ul Tropfen einer Mischung Ziegen-Anti-CTB (1: 3000) enthält, und ein Antikörper gegen ein NMDAR-Untereinheit (anti-rabbit GluN2A 1.50 , GluN2B 1:30) oder ein Anti-Kaninchen GluA2 / 3 (1:30)in TBS-Triton (TBST, 0,01% Triton X-100, pH 7,6) mit 2% BSA und 0,02 M NaN &amp; sub3; O / N bei RT. </li><li> Wash Gitter auf drei getrennte Tropfen (20 ul) von TBS (pH 7,6) für 10, 10 und 20 min, gefolgt von TBS (pH 8,2) für 5 min. </li><li> Inkubieren Gitter für 2 h auf Tropfen (20 ul) einer Mischung Esel-anti-Kaninchen-IgG (1:20), der mit 10 nm Goldpartikel und Esel anti-Ziege IgG (1:20), der mit 18 nm Goldpartikel in TBST enthaltend (pH 8,2) mit 2% BSA und 0,02 M NaN 3. </li><li> Waschen Sie Netze in 20 ul Tropfen TBS (pH 7,6) für 5, 5 und 10 Minuten, dann in ultrareinem Wasser und trocknen Sie sie dann. </li><li> Gegenfärbung Gitter mit 5% Uranylacetat und 0,3% Blei Citrat in destilliertem H 2 O 8 und 5 min bei schlechten Lichtverhältnissen auf. </li><li> Für Dreifachmarkierungsexperimente, inkubieren Gitter O / N bei RT mit 20 ul Tropfen einer Mischung von Anti-Ziege-CTB (1: 3000), anti-Maus-PSD-95 (1: 100) und anti-Kaninchen GluN2A (1 : 50). Dann brüten Gitter für 2 Stunden auf 20 ul Tropfen einer Mischung von IgGs gekoppelt bis 18, 10 und 5 nm Goldpartikel in TBST (pH 8,2) mit 2% BSA und 0,02 M NaN 3. Halten Sie die gleichen Verfahren wie Doppelmarkierung zum Waschen und Gegenfärbung zwischen und nach Antikörper-Inkubation. </li><li> Die Kontrollen wurden in dem die sekundären Antikörpern durchgeführt wurden allein aufgebracht. </li><li> Ansicht Gitter auf einem EM und digitalisieren Bilder. Prozess endgültigen Zahlen in Adobe Photoshop 6.0 ​​nur für Helligkeit und Kontrast, wenn es notwendig ist 24. </li></ol> 4. Quantifizierung <ol><li> Zur manuellen Einstellung der Bereiche der inneren plexiformen Schicht (IPL), ohne Löcher oder Risse an 8,000X Vergrößerung, dann Fotomontage zufällig die volle Tiefe von IPL in 25,000X Vergrößerung. </li><li> Identifizieren RGC Dendriten an Konus bipolaren Dyaden, wenn sie a) enthalten die retrograd transportiert CTB Signal (große Goldpartikel), b) zeigen wohldefinierte Membranen, Spalten und postsynaptischen Dichte, c) mindestens zwei kleine Gold Teil enthaltenicles innerhalb des PSD, oder mehr als eine kleine Goldpartikel entlang der extrasynaptischen Plasmamembran. </li><li> Sammeln Sie 45 - 55 RGC Dendriten, basierend auf obigen Kriterien, für die quantitative Analyse. </li><li> Messen der Längen der PSD und dem extrasynaptischen Plasmamembran von einzelnen RGC Dendriten mit NIH ImageJ Software. Graf Goldpartikel innerhalb von 10 nm der Membran als Membran-assoziierte, basierend auf einer durchschnittlichen Dicke von 7-9 nm für die Plasmamembran 26. </li><li> Berechnen Partikeldichte als die Anzahl von Goldpartikeln pro linearem Mikrometer (gold / um). Den Abstand zwischen der Mitte jedes Goldpartikel und der Mitte des PSD (für synaptische Ort) oder den Rand des PSD (für extrasynaptischen location). </li><li> Führen Sie eine statistische Analyse von Software. Verwenden zweiseitigen t-Tests Mittel zu vergleichen. Folgern Bedeutung, wenn P &lt;0,05. Wenn nicht anders angegeben, Bericht Werte als Mittelwert ± SEM 18. </li></ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metabotropic+glutamate+receptor+(mGluR1)+is+concentrated+at+perisynaptic+membrane+of+neuronal+subpopulations+as+detected+by+immunogold+reaction.">The metabotropic glutamate receptor (mGluR1) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction.</a> Neuron. 11, 771-787 (1993).</li><li>Van Lookeren Campagne, M., Oestreicher, A. B., van der Krift, T. P., Gispen, W. H., Verkleij, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freeze-substitution+and+Lowicryl+HM20+embedding+of+fixed+rat+brain:+Suitability+for+immunogold+ultrastructural+localization+of+neural+antigens.">Freeze-substitution and Lowicryl HM20 embedding of fixed rat brain: Suitability for immunogold ultrastructural localization of neural antigens.</a> J. Hist. Cyt. 39, 1267-1279 (1991).</li><li>Matsubara, A., Laake, J. H., Davanger, S., Usami, S., Ottersen, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+AMPA+receptor+subunits+at+a+glutamate+synapse:+A+quantitative+immunogold+analysis+of+hair+cell+synapses+in+the+rat+organ+of+Corti.">Organization of AMPA receptor subunits at a glutamate synapse: A quantitative immunogold analysis of hair cell synapses in the rat organ of Corti.</a> J. Neurosci. 16, 4457-4467 (1996).</li><li>Petralia, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+NMDA+receptors+at+extrasynaptic+locations.">Organization of NMDA receptors at extrasynaptic locations.</a> Neuroscience. 167, 68-87 (2010).</li><li>Merighi, A., Polak, J. M., Priestley, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-embedding+electron+microscopic+immunocytochemistry.">Post-embedding electron microscopic immunocytochemistry.</a> Electron Microscopic Immunocytochemistry: Principles and Practice. , 51-87 (1992).</li><li>Ottersen, O. P., Takumi, Y., Matsubara, A., Landsend, A. S., Laake, J. H., Usami, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+organization+of+a+type+of+peripheral+glutamate+synapse:+the+afferent+synapses+of+hair+cells+in+the+inner+ear.">Molecular organization of a type of peripheral glutamate synapse: the afferent synapses of hair cells in the inner ear.</a> Prog. Neurobiol. 54, 127-148 (1998).</li><li>Nusser, Z., Lujan, R., Laube, G., Roberts, J. D., Molnar, E., Somogyi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type+and+pathway+dependence+of+synaptic+AMPA+receptor+number+and+variability+in+the+hippocampus.">Cell type and pathway dependence of synaptic AMPA receptor number and variability in the hippocampus.</a> Neuron. 21, 545-559 (1998).</li><li>Takumi, Y., Ramirez-Leon, V., Laake, P., Rinvik, E., Ottersen, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+modes+of+expression+of+AMPA+and+NMDA+receptors+in+hippocampal+synapses.">Different modes of expression of AMPA and NMDA receptors in hippocampal synapses.</a> Nat. Neurosci. 2, 618-624 (1999).</li><li>Grimes, W. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+inhibitory+microcircuitry+regulates+retinal+signaling+near+visual+threshold.">Complex inhibitory microcircuitry regulates retinal signaling near visual threshold.</a> J. Neurophysiol. 114, 341-353 (2015).</li><li>Sassoe-Pognetto, M., Ottersen, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+ionotropic+glutamate+receptors+at+dendrodendritic+synapses+in+the+rat+olfactory+bulb.">Organization of ionotropic glutamate receptors at dendrodendritic synapses in the rat olfactory bulb.</a> J. Neurosci. 20, 2192-2201 (2000).</li></ol>

The authors have no disclosures.

Wir haben vier beschriebenen Techniken für eine erfolgreiche quantitative Post-Einbettung Immun EM: 1) kurze und schwache Fixierung, 2) Gefriersubstitution, 3) nach dem Einbetten von Immunfärbung und 4) Quantifizierung. EM Immunogold ermöglicht den Nachweis von spezifischen Proteinen in ultradünne Gewebeschnitten. Markierte Antikörper mit Goldpartikeln kann direkt unter Verwendung von EM visualisiert werden. Während mächtig in die subsynaptischen Lokalisierung eines Membranrezeptors z...

Glutamat ist der hauptsächliche exzitatorische Neurotransmitter im Netzhaut 1. Retinalen Ganglienzellen (RGC), glutamatergen synaptischen Input von bipolaren Zellen 2 sind die Ausgangsneuronen der Netzhaut erhalten, die visuelle Information an das Gehirn senden. Physiologische Untersuchungen zeigten, dass die synaptische Erregung von RGCs vermittelt wird postsynaptisch von NMDA-Rezeptoren (NMDARs) und AMPA-Rezeptoren (AMPARs) 3,4,5. Obwohl exzitatorischen postsynaptischen Ströme (EPSCs) in RGCs durch AMPARs und NMDARs 3,5,6,7,8, zeigen spontane Miniatur EPSCs (mEPSCs) auf RGCs vermittelt werden nur eine AMPARs vermittelte Komponente 4,5,9. Allerdings ergab Glutamataufnahme Reduktion eines NMDAR Komponente in spontanen EPSCs 5, was darauf hindeutet, dass NMDARs auf RGC Dendriten können außerhalb von Synapsen befinden. Membranassoziierten Guanylat-Kinasen (MAGUKs) wie PSD-95, dass Cluster Neurotransmitter-Rezeptoren, einschließlich Glutamat-Rezeptoren und Ionenkanals bei synaptischen Websites, auch deutliche subsynaptischen Expressionsmuster 10,11,12,13,14 aufweisen. In den letzten Jahrzehnten konfokalen Immunhistochemie und Pre-Einbettung Elektronenmikroskop (EM) Immunhistochemie wurden zu studieren Membran-Rezeptor-Expression eingesetzt. Obwohl konfokalen Immunfärbung breites Muster der Rezeptorexpression offenbart, macht seine geringere Auflösung unmöglich subzellulären Ort zu unterscheiden zu können. Pre-Einbettung EM-Studien in Säugernetzhaut zeigen, dass NMDAR Untereinheiten in postsynaptischen Elemente bei Konus Bipolarzelle Band Synapsen 15,16,17 vorhanden sind. Dies ist in der scheinbaren Gegensatz zu physiologischen Beweise. Jedoch Diffusion von Reaktionsprodukt ist ein bekannter Artefakt in der Pre-Einbettungs Immunperoxidase-Verfahren. Daher ist dieser Ansatz nicht in der Regel statistisch verlässliche Daten geben und Unterscheidung zwischen Lokalisierung zu synaptischen Membran gegen extrasynaptischen Membran 18,19,20,21 ausschließen. Aufdie andere Hand, physiologischen und anatomischen Daten sind mit einem synaptischen Lokalisation von AMPARs auf RGCs 3,5,7,9,22 konsistent. Somit Glutamatrezeptoren und MAGUKs bei Netzhautband Synapse nicht nur lokalisiert der postsynaptischen sondern auch auf die perisynaptic oder extrasynaptischen Membran Fächern. Jedoch ist ein hochauflösender quantitative Analyse dieser Membranproteine ​​in einer Netzhautband Synapse wird noch benötigt. Hier entwickelten wir ein postembedding EM Immunogold Technik die subsynaptischen Lokalisierung von NMDAR-Untereinheiten, AMPAR Untereinheiten und PSD-95, gefolgt von der Schätzung der Anzahl, Dichte und die Variabilität dieser Proteine ​​an Synapsen auf Ratten RGCs untersucht markierten Choleratoxin-Untereinheit B mit (CTB) Rückverfolgung Methoden.

<table><tbody><tr><td>Paraformaldehyde</td><td>EMS</td><td>15710</td><td></td></tr><tr><td>Glutaraldehyde</td><td>EMS</td><td>16019</td><td></td></tr><tr><td>NaH2PO4</td><td>Sigma</td><td>S9638</td><td></td></tr><tr><td>Na2HPO4</td><td>Sigma</td><td>7782-85-6</td><td></td></tr><tr><td>CaCl2</td><td>Sigma</td><td>C-8106</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A-7030</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T-8787</td><td></td></tr><tr><td>NaOH</td><td>Sigma</td><td>221465</td><td></td></tr><tr><td>NaN3</td><td>JT Baker</td><td>V015-05</td><td></td></tr><tr><td>Glycerol</td><td>Gibco BRL</td><td>15514-011</td><td></td></tr><tr><td>Lowicryl HM 20</td><td>Polysciences</td><td>15924-1</td><td></td></tr><tr><td>Tris-Base</td><td>Fisher</td><td>BP151-500</td><td></td></tr><tr><td>Tris</td><td>Fisher</td><td>04997-100</td><td></td></tr><tr><td>Anti-GluN2A</td><td>Millipore</td><td>AB1555P</td><td>Dilution 1/50</td></tr><tr><td>Anti-GluN2B</td><td>Millipore</td><td>AB1557P</td><td>Dilution 1/30</td></tr><tr><td>Anti-GluA2/3</td><td>Millipore</td><td>AB1506</td><td>Dilution 1/30</td></tr><tr><td>Anti-PSD-95</td><td>Millipore</td><td>MA1&amp;ndash;046</td><td>Dilution 1/100</td></tr><tr><td>Donkey anti-rabbit IgG-10 nm gold particles</td><td>EMS</td><td>25704</td><td>Dilution 1/20</td></tr><tr><td>Donkey anti-mouse IgG-10 nm gold particles</td><td>EMS</td><td>25814</td><td>Dilution 1/20</td></tr><tr><td>Donkey anti-mouse IgG-5 nm gold particles</td><td>EMS</td><td>25812</td><td>Dilution 1/20</td></tr><tr><td>Donkey anti-goat IgG-18 nm gold particles</td><td>Jackson ImmunoResearch</td><td>705-215-147</td><td>Dilution 1/20</td></tr><tr><td>Formvar-Carbon coated nickel-slot grids.</td><td>EMS</td><td>FCF2010-Ni</td><td></td></tr><tr><td>Uranyl acetate</td><td>EMS</td><td>22400-1</td><td></td></tr><tr><td>Methanol</td><td>EMS</td><td>67-56-1</td><td></td></tr><tr><td>Lead citrate</td><td>Leica</td><td></td><td></td></tr><tr><td>Leica EM AFS</td><td>Leica</td><td></td><td></td></tr><tr><td>Leica EM CPC</td><td>Leica</td><td></td><td></td></tr><tr><td>Ultramicrotome</td><td>Leica</td><td></td><td></td></tr><tr><td>JEOL 1200 EM</td><td>JEOL</td><td></td><td></td></tr><tr><td>liquid nitrogen&amp;nbsp;</td><td>Roberts Oxygen</td><td></td><td></td></tr><tr><td>Propane</td><td>Roberts Oxygen</td><td></td><td></td></tr><tr><td>CTB</td><td>List Biological Laboratories</td><td>104</td><td>1 - 1.2%</td></tr><tr><td>Anti-CTB</td><td>List Biological Laboratories</td><td>703</td><td>Dilution 1/4,000</td></tr></tbody></table>

high-resolution quantitative immunogold analysis of membrane receptors at retinal ribbon synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

Die hier vorgestellten Ergebnisse zeigen deutlich unterschiedliche subsynaptischen Lokalisierungsmuster GluA 2/3 und NMDARs auf RGC Dendriten in Rattennetzhaut, wie zuvor 24,25 beschrieben. 77% der GluA 2/3 Immunogold Partikel in RGC dendritischen Profile wurden in der PSD (1A) befindet, ähnlich wie die meisten zentralen Synapsen. Allerdings wurden NMDARs sich entweder synaptisch oder extrasynaptically. 83% der GluN2A Immunogold Teilchen wurden in dem PSD lok...

Watch this Scientific Journal Video about High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses at JoVE.com

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

Hochauflösende Quantitative Immungold-Analyse von Membranrezeptoren auf der Netzhaut Ribbon Synapsen

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

high-resolution-quantitative-immunogold-analysis-membrane-receptors

Research

JoVE Journal

Neuroscience

7.5K Aufrufe.  National Institutes of Health. The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

Serie: High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

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

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 imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.

This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.

Die Quantifizierung der Synapsen: eine Immunzytochemie-basierten Assay zur Synapse Anzahl Quantify

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 ...

Forschung

Neurowissenschaften

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43&reg;) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

Imaging Exocytose in Retinal bipolaren Zellen mit TIRF-Mikroskopie

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

Biologie

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

Post-Einbettung Immunogoldmarkierung der synaptischen Proteine ​​in Hippocampusschnitt Kulturen

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

Immunhistochemischen und Calcium Imaging Methoden in der Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).
The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre- and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.
Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning.

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

Kombinierte optogenetische und Gefrierbruch Replica Immunmarkierung Eingang spezifische Anordnung der Glutamate Receptors in der Maus Amygdala zu untersuchen

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

Imaging the Human Immunological Synapse

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90&#176; angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS.

This protocol images both the immunological synapse formation and the subsequent polarized secretory traffic towards the immunological synapse. Cellular conjugates were formed between a superantigen-pulsed Raji cell (acting as an antigen-presenting cell) and a Jurkat clone (acting as an effector helper T lymphocyte).

Bildgebung der humanen immunologischen Synapse

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) ...

Immunologie und Infektion

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

<meta charset="utf8"></head><body>Optimiertes Protokoll für die Vorbereitung von retinalen Organoid-Proben für TEM

Preparing Retinal Organoid Samples for Transmission Electron Microscopy

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem cells (iPSCs) under specific conditions. Synapse development and maturation in ROs have been studied immunocytochemically and functionally. However, the direct evidence of the synaptic contact ultrastructure is limited, containing both special ribbon synapses and conventional chemical synapses. Transmission electron microscopy (TEM) is characterized by high resolution and a respectable history elucidating retinal development and synapse maturation in humans and various species. It is a powerful tool to explore synaptic structure in ROs and is widely used in the research field of ROs. Therefore, to better explore the structure of RO synaptic contacts at the nanoscale and obtain high-quality microscopic evidence, we developed a simple and repeatable method of RO TEM sample preparation. This paper describes the protocol, reagents used, and detailed steps, including RO fixation preparation, post fixation, embedding, and visualization.

<meta charset="utf8"></head><body>Autor im Rampenlicht: Analyse der synaptischen Ultrastruktur in reifen retinalen Organoiden mit TEM

This protocol provides an optimized and elaborate preparation procedure for retinal organoid samples for transmission electron microscopy. It is suitable for applications that involve the analysis of synapses in mature retinal organoids.

Vorbereitung von retinalen Organoidproben für die Transmissionselektronenmikroskopie

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem ...

Hochauflösende Quantitative Immungold-Analyse von Membranrezeptoren auf der Netzhaut Ribbon Synapsen

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Kapitel in diesem Video

Die Quantifizierung der Synapsen: eine Immunzytochemie-basierten Assay zur Synapse Anzahl Quantify

Imaging Exocytose in Retinal bipolaren Zellen mit TIRF-Mikroskopie

Post-Einbettung Immunogoldmarkierung der synaptischen Proteine in Hippocampusschnitt Kulturen

Immunhistochemischen und Calcium Imaging Methoden in der Wholemount Rat Retina

Kombinierte optogenetische und Gefrierbruch Replica Immunmarkierung Eingang spezifische Anordnung der Glutamate Receptors in der Maus Amygdala zu untersuchen

Bildgebung der humanen immunologischen Synapse

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Vorbereitung von retinalen Organoidproben für die Transmissionselektronenmikroskopie

Vorbereitung von retinalen Organoidproben für die Transmissionselektronenmikroskopie

Vorbereitung von retinalen Organoidproben für die Transmissionselektronenmikroskopie

Hochauflösende Quantitative Immungold-Analyse von Membranrezeptoren auf der Netzhaut Ribbon Synapsen

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Die Quantifizierung der Synapsen: eine Immunzytochemie-basierten Assay zur Synapse Anzahl Quantify

Imaging Exocytose in Retinal bipolaren Zellen mit TIRF-Mikroskopie

Post-Einbettung Immunogoldmarkierung der synaptischen Proteine ​​in Hippocampusschnitt Kulturen

Immunhistochemischen und Calcium Imaging Methoden in der Wholemount Rat Retina

Kombinierte optogenetische und Gefrierbruch Replica Immunmarkierung Eingang spezifische Anordnung der Glutamate Receptors in der Maus Amygdala zu untersuchen

Bildgebung der humanen immunologischen Synapse

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Vorbereitung von retinalen Organoidproben für die Transmissionselektronenmikroskopie

Vorbereitung von retinalen Organoidproben für die Transmissionselektronenmikroskopie

Vorbereitung von retinalen Organoidproben für die Transmissionselektronenmikroskopie

Post-Einbettung Immunogoldmarkierung der synaptischen Proteine in Hippocampusschnitt Kulturen