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>

<ol>
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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 border="0" cellpadding="0" cellspacing="0" width="761">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Paraformaldehyde</td>
			<td style="width:167px;">EMS</td>
			<td style="width:127px;">15710</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glutarldehyde</td>
			<td>EMS</td>
			<td>16019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaH2PO4</td>
			<td>Sigma</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</td>
			<td>Sigma</td>
			<td style="width:127px;">7782-85-6</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2</td>
			<td>Sigma</td>
			<td>C-8106</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>Sigma</td>
			<td>A-7030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T-8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaOH</td>
			<td>Sigma</td>
			<td>221465</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaN3</td>
			<td>JT Baker</td>
			<td>V015-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Gibco BRL</td>
			<td>15514-011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lowicryl HM 20</td>
			<td>Polysciences</td>
			<td>15924-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-Base</td>
			<td>Fisher</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris</td>
			<td>Fisher</td>
			<td>04997-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-GluN2A</td>
			<td>Millipore</td>
			<td>AB1555P</td>
			<td>Dilution 1/50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-GluN2B</td>
			<td>Millipore</td>
			<td>AB1557P</td>
			<td>Dilution 1/30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-GluA2/3</td>
			<td>Millipore</td>
			<td>AB1506</td>
			<td>Dilution 1/30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-PSD-95</td>
			<td>Millipore</td>
			<td>MA1&#8211;046</td>
			<td>Dilution 1/100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donkey anti-rabbit IgG-10 nm gold particles</td>
			<td>EMS</td>
			<td>25704</td>
			<td>Dilution 1/20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donkey anti-mouse IgG-10 nm gold particles</td>
			<td>EMS</td>
			<td>25814</td>
			<td>Dilution 1/20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donkey anti-mouse IgG-5 nm gold particles</td>
			<td>EMS</td>
			<td>25812</td>
			<td>Dilution 1/20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donkey anti-goat IgG-18 nm gold particles</td>
			<td>Jackson ImmunoResearch</td>
			<td style="width:127px;">705-215-147</td>
			<td>Dilution 1/20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formvar-Carbon coated nickel-slot grids.</td>
			<td>EMS</td>
			<td>FCF2010-Ni</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Uranyl acetate</td>
			<td>EMS</td>
			<td>22400-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>EMS</td>
			<td>67-56-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lead citrate</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica EM AFS</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica EM CPC</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultromicrotome</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">JEOL 1200 EM</td>
			<td>JEOL</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">liquid nitrogen&#160;</td>
			<td>Roberts Oxygen</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propane</td>
			<td>Roberts Oxygen</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CTB</td>
			<td>List Biological Laboratories</td>
			<td>104</td>
			<td>1-1.2%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CTB</td>
			<td>List Biological Laboratories</td>
			<td>703</td>
			<td>Dilution 1/4000</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

7.4K 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

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

Automatisierte Sholl Analyse digitalisierter neuronale Morphologie bei Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

Forschung

Neurowissenschaften

Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the postsynaptic activity of a single ribbon synapse 1-4. Ribbon synapses of sensory cells release neurotransmitter continuously, the rate of which is modulated in response to graded changes in IHC membrane potential 5. Ribbon synapses have been shown to operate by multivesicular release, where multiple vesicles can be released simultaneously to evoke excitatory postsynaptic currents (EPSCs) of varying amplitudes 1, 4, 6-11. Neither the role of the presynaptic ribbon, nor the mechanism underlying multivesicular release is currently well understood.
The IHC is innervated by 10-20 auditory nerve fibers, and every fiber contacts the IHC with a unmyelinated single ending to form a single ribbon synapse. The small size of the afferent boutons contacting IHCs (approximately 1 &mu;m in diameter) enables recordings with exceptional temporal resolution to be made. Furthermore, the technique can be adapted to record from both pre- and postsynaptic cells simultaneously, allowing the transfer function at the synapse to be studied directly 2. This method therefore provides a means by which fundamental aspects of neurotransmission can be studied, from multivesicular release to the elusive function of the ribbon in sensory cells.

Whole-cell patch-clamp recordings from auditory nerve fiber dendrites at the inner hair cell ribbon synapse in the mammalian cochlea.

Postsynaptische Recordings am afferenten Dendriten Kontaktaufnahme mit Cochlear inneren Haarzellen: Monitoring multivesikulären Veröffentlichung in einem Ribbon Synapse

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

Präparation einer Maus Eye für einen ganzen Berg von retinalen Pigmentepithel

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

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.

Quantitative Analyse der synaptischen Vesikel Pool Replenishment in Cultured Cerebellar Nervenzellen Granulat mit FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

Hochauflösende Functional Magnetic Resonance Imaging Methods for Human Mittelhirn

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

Hochauflösende Live-Bildgebung von Zell-Verhalten in der Entwicklung von Neuroepithel

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

Dissection, Kultur und Analyse der<em&gt; Xenopus laevis</em&gt; Embryonic Netzhautgewebe

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors&#8212;an essential step toward a model of odor coding in the mammalian olfactory system.

Olfactory receptor activation patterns encode odor identity, but the lack of published data identifying odorant ligands for mammalian olfactory receptors hinders the development of a comprehensive model of odor coding. This protocol describes a method to facilitate high-throughput identification of olfactory receptor ligands and quantification of receptor activation.

Hochdurchsatz-Analyse von Säugetiergeruchsrezeptoren: Die Messung der Rezeptoraktivierung über Luciferaseaktivität

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors ...

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

Metabolomische Analyse von Rattenhirn Hohe Auflösung Kernresonanzspektroskopie von Gewebeextrakte

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics ...

Peering at Brain Polysomes with Atomic Force Microscopy

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

Peering am Gehirn Polysomen mit Rasterkraftmikroskopie

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...