This work was supported by NIH grants (NS070577 and NS084869).

Hinweis: Die Zusammensetzung aller nachstehend genannten Lösungen können in der Werkstoffe / Zubehör Tabelle gefunden werden. 1. Protein A Säule Reinigung der Antikörper <ol><li> Äquilibrieren ein Protein, eine Spin-Säule und alle benötigten Puffer auf Raumtemperatur 15 min vor dem Beginn des Verfahrens. </li><li> Verdünne das Immunserum 1: 1 mit Protein A IgG Bindungspuffer. (5 ml Serum wird empfohlen). </li><li> Klopfen Sie leicht ein Protein, eine Spin-Säule auf der Tischplatte, jede Harz, das in die Kappe eingelegt werden können, zu entfernen. Entfernen Sie die obere Kappe und sanft Snap aus dem Bodenverschluss. Legen Sie die Spalte in einem 15 ml-Collection-Tube und lassen Speicherlösung ablaufen. </li><li> 5 ml Protein A IgG Bindungspuffer und die Lösung ablaufen. </li><li> Übernehmen Sie die verdünnt Immunserum an die Säule und sammeln Sie die Durchströmung. Für beste Ergebnisse, einen Probenvolumen, das eine Antikörperkonzentration als 80% der Antikörper-bindenden capacit der Spalte enthält wenigery. </li><li> Mit 15 ml Protein A-IgG-Bindungspuffer oder bis die Absorption bei 280 nm unter 0,1 Die Säule. </li><li> 100 l Neutralization Buffer zu fünf markierten Sammelröhrchen. </li><li> 5 ml der IgG Elutionspuffer auf Protein A-Säule und Sammeln Fraktionen von 1 ml in jedes der Rohre mit den 100 ul Neutralisierungspuffer. </li><li> Die Extinktion jeder Fraktion bei 280 nm und Poolfraktionen mit einer Absorption von mehr als 0,5. Halten Sie die vereinigten Fraktionen auf Eis, bis sie zur Dialyse. </li><li> Regenerieren die Säule durch Zugabe von 8 ml IgG Elution Buffer und lassen Sie die Lösung durch die Säule fließen. </li><li> Spülen der Säule mit Protein A IgG Bindungslösung, bis die Laufmittel pH wieder auf 7,4. </li><li> Bewahren Sie die Spalte durch Zugabe von 5 ml-Speicherlösung (0,02% Natriumazid in PBS). Wenn etwa 3 ml bleiben in der Spalte, Kappenboden und befestigen Sie die obere Abdeckung. Bewahren Sie die Spalte bei 4 ° C. </li><li> Bestimmen Sie die Länge of Dialyseschlauch nach der Anleitung des Herstellers erforderlich ist. Unter Verwendung von 16 mm Flachrohrdurchmesser verwenden 5.47 cm Rohrlänge pro ml Lösung, um dialysiert werden. Schneiden Sie den Schlauch und Spülen mit PBS, pH 7,4, für 15 bis 30 min. </li><li> Falten und Clip ein Ende der Dialyseschlauch, überweisen Sie den gepoolten IgG enthaltenden Fraktionen in einen Dialyseschlauch in PBS pH 7,4 ins Gleichgewicht gebracht, und schließen Sie das andere Ende. </li><li> Dialyse bei Raumtemperatur gegen 200 Volumina PBS pH 7,4 für 2 Stunden. </li><li> Ändern die Dialysepuffer (frisch PBS pH 7,4) dialysiert und für weitere 2 Stunden bei Raumtemperatur. </li><li> Ändern Sie den Dialysepuffer (frisches PBS pH 7,4) dialysiert und über Nacht bei 4 ° C. </li><li> Halten Sie die dialysiert Antikörperlösung bei 4 ° C, bis bereit, mit weiteren Reinigungsschritten fortfahren. </li><li> Bewertung der Antikörperkonzentration unter Verwendung seiner Absorption bei 280 nm und der molare Absorptionskoeffizient von Antikörpern bei 280 nm von 210.000 M -1 cm -1 12. </li></ol> 2. Kupplung von LBD Proteine, um die Kopplung Resin Aminolink <ol><li> Bereiten Sie zwei Spalten, eine für Nor1 und die andere für Nur77. Folgen Sie dem gleichen Protokoll sowohl Proteinen. </li><li> Aufzutauen, dass das Protein an das Harz auf Eis gekoppelt werden. Bestimmung der Proteinkonzentration unter Verwendung seines molaren Extinktionskoeffizienten und seine Absorption bei 280 nm 12. <ol><li> Alternativ können Sie einen Proteinbestimmung Assay wie dem Bicinchoninsäure-Assay 13. Wenn das Protein in einer aminhaltigen Puffer dialysiert oder Pufferaustausch gegen den Kopplungspuffer gelöst. Wenn das Protein in einem geeigneten Puffer (keine freien Amine) verdünnen 4fach in Kopplungspuffer. </li></ol></li><li> Ein 2-ml Harz für 2 mg Protein. Alle Schritte, die folgen, beziehen sich auf 2 ml Harz in einem 10-ml-Säule. Bis zu 10 mg Protein pro 1 ml Harz (: 1 Aufschlämmung 2 ml 1) verbunden werden. </li><li> Bereiten Sie eine 10 ml-Säule, indem Sie eine Fritte nach unten und läuft PBS pH 7,4 durch. Cap ter Boden der Säule und 2 ml Kopplungspuffer. </li><li> Fügen Sie das gewünschte Volumen an Schlamm (4 ml bis 2 ml Harz zu erhalten) an die Säule, entfernen Sie die Bodenkappe und lassen Sie die Spalte Drain. Spülen der Säule mit einer Gesamtzahl von 6 ml (3 Harz-Bettvolumen) Kopplungspuffer und damit der Säule ablaufen. Nach der Kupplungspuffer entleert, ersetzen Sie die Bodenkappe. </li><li> Hinzuzufügen 2-4 ml Protein in Kupplungspuffer suspendiert, um die Säule (keep ein Aliquot der Proteinlösung, um die Kopplungseffizienz, indem die Proteinkonzentration des Eluats in Schritt 2.11 unter Verwendung von entweder der Absorption bei 280 nm oder die Bicinchoninsäure-Assay zu beurteilen) . Kappe und mischen kopfüber, um eine homogene Lösung zu haben. </li><li> Wiegen Sie eine leere 1,8 ml Mikrozentrifugenröhrchen und zu bewegen, um den Abzug. Sorgfältig über NaCNBH 3 (etwa 0,05 g) in der vorgewogen Rohr. Schließen Sie den Schlauch und wiegen die Röhrchen mit NaCNBH 3. Das Rohr Öffnen Sie nicht außerhalb der Haube. </li><li> Bezogen auf das GewichtNaCNBH 3 in das Rohr übertragen, machen Sie eine 5 M-Lösung durch Zugabe von 1 M NaOH. Das Molekulargewicht des NaCNBH 3 62,84 g deshalb 0,05 g gleich (0,05 / 62,84 = 7,96 x 10 -4 Mol) 7,96 x 10 -4 mol ist. Um eine 5 M Lösung add (7,96 x 10 -4 / 5) 159 ul 1 M NaOH zu machen. </li><li> Messen Sie Gesamtvolumen der Reaktion der Einnahme des Harzvolumen in Betracht. In 10 ul 5 M NaCNBH 3 pro ml Reaktions. <ol><li> Zum Beispiel: für einen 4 ml Reaktionsvolumen werden 40 &amp; mgr; l von 5 M NaCNBH 3. 1 ml Harz und 3 ml zugesetztes Protein, das Gesamtvolumen von 4 ml. </li></ol></li><li> Verschließen Sie die Spalte und Mischen über Kopf. Befestigen Sie die Säule an einem Rohr Rotator und ließ die Reaktion über Nacht weiter bei 4 ° C. </li><li> Am Morgen bringen die Spalte an die chemischen Haube und entfernen Sie die Kappe so etwas Gas gebildet haben sorgfältig. Entleeren Sie die Säule in ein sauberes Röhrchen und speichern Sie das Eluat, das Protein con messentration mit der Absorption bei 280 nm Methode 12 oder die Bicinchoninsäure-Assay und vergleichen ihn mit dem Ausgangsprotein-Konzentration in Schritt 2.6. </li><li> Das Harz wird mit 4 ml Kopplungspuffer und lassen Sie die Spalte Drain. </li></ol> 3. Blocking Verbleibende Seiten <ol><li> Mit 4 ml Puffer Abschrecken Waschen des Harzes. Abtropfen lassen und ersetzen Sie die Bodenkappe. 2 ml Puffer und Abschrecken auszusetzen Harzes. </li><li> Beurteilung des gesamten Reaktionsvolumens durch Messung der Harzvolumen und das Abschrecken Puffervolumen dann werden 10 ul 5 M NaCNBH 3 pro ml Reaktionsvolumen. </li><li> Vorsichtig mischen bei Raumtemperatur für 30 min End-Over-End. Nach 30 Minuten bringen Sie die Spalte in der Abzugshaube. Entfernen Sie vorsichtig die obere und dann die untere Kappe und lassen Sie die Spalte Drain in einen Abfallrohr. </li><li> Mit 5 Harzvolumina Waschlösung Die Säule. Überwachen Sie die Waschanlagen für die Anwesenheit von Restproteine ​​durch Messung des Eluats der absorbance bei 280 nm. Entkoppelt Proteine ​​werden durch die hohe Salzkonzentration gewaschen. </li><li> Mit 6 ml entgastem PBS pH 7,4, enthaltend 0,02% Natriumazid Waschen des Harzes. Halten der Säule auf 4 ° C, bis es benötigt. </li></ol> 4. Reinigung von Nurr1 spezifischer Antikörper <ol><li> Spülen Sie die Nur77 LBD gekoppelt Säule mit 10 ml PBS pH 7,4 und lassen Sie die Spalte Drain. Verschließen Sie die unteren Ende der Säule und legen Sie das abgelassen Nur77 LBD gekoppelt Säule in ein neues 15 ml Sammelröhrchen. </li><li> 5 ml Protein A-gereinigten Anti-Nurr1-Antikörpern, die Kappe die Säule und den Platz auf einem Schüttler bei 4 ° C für 1 Stunde. Entfernen Sie die oberen und unteren Kappen und sammeln die Strömung durch. Beiseite auf Eis, bis bereit gesetzt, um zum Nor1 LBD gekoppelt Spalte hinzuzufügen. </li><li> Spülen Sie die Nor1 LBD gekoppelt Säule mit 10 ml PBS pH 7,4 und lassen Sie die Spalte Drain. Verschließen Sie die unteren Ende der Säule und legen Sie die Nor1 LBD gekoppelt Spalte in einem 15 ml-Collection-Tube. </li><li> In den Fluss durch von der Nur77 Putsch LBDLED-Säule vom Säule und auf einem Rotator bei 4 ° C für 1 Stunde. Entfernen Sie die oberen und unteren Kappen und sammeln die Strömung durch. </li><li> Messung der Antikörperkonzentration durch Messung der Absorption bei 280 nm. </li></ol> 5. ELISA-basierte Analyse von gereinigten Anti-Nurr1 Antikörper <ol><li> 100 l Protein (2,5 ug / ml) in die Vertiefungen einer 96 Well-Platte. Wenn die Prüfung 3 verschiedene Proteine ​​wird mit 100 ul Protein 1 die Vertiefungen A1 bis H4 wurden 100 ul Protein 2, die Vertiefungen A5 bis H8 und 100 &amp; mgr; l von Protein 3 zu Vertiefungen A9 bis H12. Die Platte und Inkubation über Nacht bei 4 ° C. </li><li> Zweimaliges Waschen der beschichteten Wells mit 300 ul pro Napf an PBS, pH 7,4 und 300 ul ELISA Blockpuffer an die gesamte Antigen-beschichteten Vertiefungen und inkubiert 2 h bei Raumtemperatur. </li><li> Während die Platte blockiert Vorbereitung eines gewünschten Ausgangsverdünnung von gereinigtem anti-Nurr1-Antikörper (im Bereich von 10 bis 100 ug / ml) im ELISA Blockpuffer. </li><li> Nach 2 Stunden der Sperrung, ersetzen Sie den ELISA-Blockpuffer mit 100 ul frisch ELISA-Blockpuffer in alle Vertiefungen. </li><li> 100 l des verdünnten gereinigten anti-Nurr1 Antikörper in jede Vertiefung in der Reihe A. Seriell verdünnte die Antikörper durch die Übertragung von 100 ul der Lösung von Reihe A zu den Vertiefungen in der Reihe B, gefolgt von Übertragen von 100 ul der Lösung von Reihe zu Reihe B C Fortsetzung auf G. rudern Nach dem Mischen der Lösungen in den Vertiefungen in der Reihe G entsorgen Sie die zusätzliche 100 ul. Wells in Reihe H erhalten nur die ersten 100 l Blocking-Puffer. Inkubieren bei Raumtemperatur für 1 Stunde. </li><li> Drei (3) mal mit 300 ul pro Vertiefung von 0,05% Tween 20 waschen Sie die Platte in PBS pH 7,4 und trocknen Sie die Platte, um überschüssigen Waschpuffer zu entfernen. </li><li> 100 l Meerrettich-Peroxidase (HRP) konjugiertem Ziegen-Anti-Kaninchen-IgG ELISA in Blockpuffer verdünnt (1: 8.000 Verdünnung; der typische Bereich von 1: 5.000 bis 1: 25.000). Inkubieren bei Raumtemperatur für 1 Stunde. </li><li> Waschen Sie die Platte threie (3) mal so in 5.6 beschrieben. Spülen Sie alle Vertiefungen durch mit 300 ul entionisiertem Wasser füllt sie. Tupfen Sie überschüssiges Wasser durch Umdrehen der Platte auf absorbierendes Papier. </li><li> Füge 100 ul 3, 3 &#39;, 5, 5&#39; Tetramethylbenzidin (auf Raumtemperatur erwärmt) in jede Vertiefung und inkubiert 15 bis 30 min im Dunkeln bei Raumtemperatur. </li><li> Stoppen Sie die Reaktion durch Zugabe von 50 ul 2 NH 2 SO 4. </li><li> Extinktion bei 450 nm subtrahiert Hintergrund bei 650 nm. </li></ol>

<ol>
	<li>Hawk, J. D., Abel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+NR4A+transcription+factors+in+memory+formation.">The role of NR4A transcription factors in memory formation.</a> Brain Res. Bull. 85 (1-2), 21-29 (2011).</li><li>Law, S. W., Conneely, O. M., DeMayo, F. J., O'Malley, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+new+brain-specific+transcription+factor,+NURR1.">Identification of a new brain-specific transcription factor, NURR1.</a> Mol. Endocrinol. 6 (12), 2129-2135 (1992).</li><li>Zetterstr&#246;m, R. H., 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=Dopamine+neuron+agenesis+in+Nurr1-deficient+mice.">Dopamine neuron agenesis in Nurr1-deficient mice.</a> Science. 276 (5310), 248-250 (1997).</li><li>Kadkhodaei, B., 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=Nurr1+is+required+for+maintenance+of+maturing+and+adult+midbrain+dopamine+neurons.">Nurr1 is required for maintenance of maturing and adult midbrain dopamine neurons.</a> J. Neurosci. 29 (50), 15923-15932 (2009).</li><li>Decressac, M., Volakakis, N., Bj&#246;rklund, A., Perlmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NURR1+in+Parkinson+disease--from+pathogenesis+to+therapeutic+potential.">NURR1 in Parkinson disease--from pathogenesis to therapeutic potential.</a> Nat Rev Neurol. 9 (11), 629-636 (2013).</li><li>Pe&#241;a de Ortiz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+Expression+of+the+Orphan+Nuclear+Receptor+Gene+hzf-3/nurr1+during+Spatial+Discrimination+Learning.">Hippocampal Expression of the Orphan Nuclear Receptor Gene hzf-3/nurr1 during Spatial Discrimination Learning.</a> Neurobiol Learn Mem. 74 (2), 161-175 (2000).</li><li>Vecsey, C. G., 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=Histone+deacetylase+inhibitors+enhance+memory+and+synaptic+plasticity+via+CREB:CBP-dependent+transcriptional+activation.">Histone deacetylase inhibitors enhance memory and synaptic plasticity via CREB:CBP-dependent transcriptional activation.</a> J. Neurosci. 27 (23), 6128-6140 (2007).</li><li>Col&#243;n-Cesario, W. I., 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=Knockdown+of+Nurr1+in+the+rat+hippocampus:+implications+to+spatial+discrimination+learning+and+memory.">Knockdown of Nurr1 in the rat hippocampus: implications to spatial discrimination learning and memory.</a> Learn Mem. 13 (6), 734-744 (2006).</li><li>McQuown, S. C., 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=HDAC3+is+a+critical+negative+regulator+of+long-term+memory+formation.">HDAC3 is a critical negative regulator of long-term memory formation.</a> J. Neurosci. 31 (2), 764-774 (2011).</li><li>Hawk, J. D., 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=NR4A+nuclear+receptors+support+memory+enhancement+by+histone+deacetylase+inhibitors.">NR4A nuclear receptors support memory enhancement by histone deacetylase inhibitors.</a> J. Clin. Invest. 122 (10), 3593-3602 (2012).</li><li>Bridi, M. S., Abel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NR4A+orphan+nuclear+receptors+mediate+transcription-dependent+hippocampal+synaptic+plasticity.">The NR4A orphan nuclear receptors mediate transcription-dependent hippocampal synaptic plasticity.</a> Neurobiol Learn Mem. 105, 151-158 (2013).</li><li>Gill, S. C., von Hippel, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calculation+of+protein+extinction+coefficients+from+amino+acid+sequence+data.">Calculation of protein extinction coefficients from amino acid sequence data.</a> Anal. Biochem. 182 (2), 319-326 (1989).</li><li>Smith, P. K., 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=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Anal. Biochem. 150 (1), 76-85 (1985).</li><li>Zetterstr&#246;m, R. H., Williams, R., Perlmann, T., Olson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+expression+of+the+immediate+early+transcription+factors+Nurr1+and+NGFI-B+suggests+a+gene+regulatory+role+in+several+brain+regions+including+the+nigrostriatal+dopamine+system.">Cellular expression of the immediate early transcription factors Nurr1 and NGFI-B suggests a gene regulatory role in several brain regions including the nigrostriatal dopamine system.</a> Brain Res. Mol. Brain Res. 41 (1-2), 111-120 (1996).</li><li>Xiao, Q., Castillo, S. O., Nikodem, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+messenger+RNAs+for+the+orphan+nuclear+receptors+Nurr1+and+Nur77+(NGFI-B)+in+adult+rat+brain+using+in+situ+hybridization.">Distribution of messenger RNAs for the orphan nuclear receptors Nurr1 and Nur77 (NGFI-B) in adult rat brain using in situ hybridization.</a> Neuroscience. 75 (1), 221-230 (1996).</li></ol>

Die Autoren haben nichts zu offenbaren.

Der Erfolg dieses Protokoll beruht auf der Verfügbarkeit von reinen Proteinen zur Charakterisierung von Antikörpern gegen ein bestimmtes Mitglied der Proteinfamilie von Interesse angehoben. Es besteht keine Notwendigkeit, um die Antikörper unter Verwendung von Protein A / G bis zum deutlich festgestellt, dass es signifikante Kreuzreaktivität mittels ELISA und Western-Blotting zu reinigen. Wie im Text erwähnt, ist es wichtig zu vermeiden, Suspendieren des Proteins (en) werden auf der Sä...

Der Transkriptionsfaktor Nurr1 und seinen Homologen (Nur77 und Nor1) gehören zu der Kernrezeptorfamilie 4A (NR4A) 1. Sie sind auch Orphanrezeptoren weil ihre endogene Liganden noch nicht identifiziert. Nurr1 wurde erstmals 1992 kloniert, und obwohl bekannt ist, im Gehirn 2 ausgedrückt werden, wurde ihre wesentliche Rolle für die Entwicklung und Wartung von Mittelhirnneuronen mit Dopamin-knockout Maus-Studien 3 offenbart. Außerdem wurde seine wichtige Rolle für die Aufrechterhaltung der Dopamin-Neuronen im Mittelhirn vor kurzem von einem Knockout-Studie 4 gezeigt. Aufgrund dieser eleganten Studien zeigen Nurr1 kritische Rollen für Dopamin-Neuronen im Mittelhirn, viele nachfolgende Untersuchungen weitgehend auf die Dopamin-Neuronen im Mittelhirn und Dopamin bezogene neurodegenerative Erkrankung konzentriert, Parkinson-Krankheit (PD) 5. Bemerkenswert ist, Nurr1 nicht nur in den Mittelhirn Dopamin (MDA) Neuronen, sondern auch in di ausgedrücktVers Gehirnbereichen 2, was darauf hindeutet, dass es möglicherweise funktionelle Rolle in vielen nicht-DA Bereichen, die stark durch neuere Studien zeigen, dass Nurr1 spielt eine wichtige Rolle in verschiedenen Hirnfunktionen unterstützt wird. Beispiele wurde wie Lern gezeigt, dass die Speicher-induzierende Aktivitäten oder andere Hippocampus abhängigen Aufgaben ergeben sich im Hochregulierung von Nurr1-Expression im Hippocampus 6,7. Darüber hinaus nach unten von Nurr1-Expression im Hippocampus Klopf war ausreichend, um das Langzeitgedächtnis und / oder der synaptischen Plastizität 8-11 beeinträchtigen, was stark darauf hinweist, daß Nurr1 spielt verschiedene Rollen in vielen Bereichen des Gehirns. Um somit die zelltypspezifisch und subzellulären Expression Nurr1 weiter zu verstehen, ist es wünschenswert, Nurr1-spezifische Antikörper, die keine Kreuzreaktivität gegenüber seinen Homologen oder Nur77 Nor1 nicht aufweisen, zu verwenden. Dieses Papier beschreibt eine Pre-Adsorption-Protokoll, um Nurr1-spezifische Antikörper vorhanden und zusätzliche Daten, die seine Spezifität zu erzeugen.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Purified Ligand Binding Domain from Nurr1, Nor 1 and Nur77</td>
			<td></td>
			<td></td>
			<td>Column Storage solution</td>
		</tr>
		<tr>
			<td>AminoLink Coupling Resin and Kit</td>
			<td>Thermo Scientific</td>
			<td>44890</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein A spin column</td>
			<td>Thermo Scientific</td>
			<td>89978</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well Flate-bottom plates, High Flange, 330&#956;L</td>
			<td>Thermo Scientific</td>
			<td>3455</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Normal Goat Serum</td>
			<td>KPL</td>
			<td>50-675-69</td>
			<td></td>
		</tr>
		<tr>
			<td>Milk Diluent/ Blocking Concentrate&#160;</td>
			<td>KPL</td>
			<td>50-82-01</td>
			<td></td>
		</tr>
		<tr>
			<td>IgG Elution Buffer</td>
			<td>Thermo Scientific</td>
			<td>21004</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure 1 M Tris-HCI Buffer, pH 7.5</td>
			<td>Life Technologies</td>
			<td>15567-&#173;&#8208;027</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23235</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Radish Peroxidase conjugated Goat anti-Rabbit IgG (H+L)</td>
			<td>Thermo Scientific</td>
			<td>31460</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Resin</td>
			<td>Thermo Scientific</td>
			<td>88221</td>
			<td></td>
		</tr>
		<tr>
			<td>3, 3&#39;, 5, 5&#39; Tetramethylbenzydine</td>
			<td>Thermo Scientific</td>
			<td>34028</td>
			<td></td>
		</tr>
		<tr>
			<td>2N H2SO4</td>
			<td>Macron Fine Chemicals</td>
			<td>H381 05</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein A IgG Binding Buffer</td>
			<td>Thermo Scientific</td>
			<td>21001</td>
			<td></td>
		</tr>
		<tr>
			<td>IgG Elution Buffer</td>
			<td>Thermo Scientific</td>
			<td>21004</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutralization Buffer</td>
			<td></td>
			<td></td>
			<td>1M Tris-&#173;&#8208;HCl pH 8.5</td>
		</tr>
		<tr>
			<td>Column Storage solution</td>
			<td></td>
			<td></td>
			<td>Phosphate Buffered Saline containing 0.02% sodium azide</td>
		</tr>
		<tr>
			<td>Spectra/Por 7 pre-treated dialysis tubing</td>
			<td>Spectrum Labs</td>
			<td>132128</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml Disposable columns</td>
			<td>Thermo Scientific</td>
			<td>29924</td>
			<td></td>
		</tr>
		<tr>
			<td>AminoLink Coupling Buffer</td>
			<td></td>
			<td></td>
			<td>0.1M sodium phosphate, 0.05% NaN3, pH 7.0</td>
		</tr>
		<tr>
			<td>Quenching buffer</td>
			<td></td>
			<td></td>
			<td>1M Tris. HCI, 0.05% NaN3, pH 
			7.4</td>
		</tr>
		<tr>
			<td>Wash Solution</td>
			<td></td>
			<td></td>
			<td>1M NaCl, 0.05% NaN3</td>
		</tr>
		<tr>
			<td>ELISA Blocking buffer</td>
			<td></td>
			<td></td>
			<td>1% Normal Horse serum (NHS), 1% Normal Goat Serum (NGS) in 1X KPL milk diluent</td>
		</tr>
		<tr>
			<td>Storage Solution</td>
			<td></td>
			<td></td>
			<td>PBS pH 7.4 containing 0.02% sodium azide</td>
		</tr>
		<tr>
			<td>Micropipettes: 10 &#956;l; 20 &#956;l; 200 &#956;l; 1000 &#956;l</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

production of nurr-1 specific polyclonal antibodies free of cross-reactivity against its close homologs, nor1 and nur77

The nuclear receptor subfamily 4 (NR4A) is composed of 3 related proteins sharing a DNA binding domain (DBD) and a ligand-binding domain (LBD). The nuclear receptor related 1 protein (Nurr1 or NR4A2) plays a key role in the maintenance of the dopaminergic system. Dopamine dysfunctions associated with the Nurr1 gene include Parkinson&#8217;s disease, schizophrenia and manic depression among others. Furthermore, recent evidence indicates that Nurr1 is also expressed in other brain areas such as the hippocampus and plays critical roles for learning and memory. The other members of the family are nerve growth factor IB (Nur77 or NR4A1) and neuron-derived orphan receptor 1 (NOR1 or NR4A3). To help investigate the precise functional roles of Nurr1 in dopaminergic and other brain region-related neuronal dysfunctions antibodies devoid of cross-reactivities against Nur77 and NOR1 were needed. Since the proteins are more divergent in their LBDs than in their DNA binding domains immunization with purified LBDs should yield antibodies specific for Nurr1 with minimal reactivities against Nur77 and/or NOR1. Although anti-Nurr1 antibodies were successfully generated these showed significant immunoreactivity against the other members of the family. Affinity chromatography over immobilized Protein A followed by pre-adsorption against immobilized Nur77 and NOR1 LBDs yielded Nurr1 specific antibodies free of cross-reactivity. Here, we selectively target antibodies against a specific member of a highly conserved family of proteins by immunizing animals with their most divergent regions followed by removing cross reactive antibodies by pre-adsorption. The goal of the protocol is to increase polyclonal antibodies specificity through pre-adsorption against cross-reactive antigens.

Ein Vergleich der Protein A-Säule gereinigt Nurr1-spezifische Antikörper mit Protein A gereinigter anti-Nurr1-Antikörper, gefolgt von Passage durch Nur77 LBD und Nor1 LBD Spalten ist in Figur 1 dar. Wie gezeigt ist ersichtlich, Protein A-gereinigten Nurr1-Antikörper zeigte eine starke Bindungs um Nurr1 LBD. Jedoch zeigte sich auch eine signifikante Bindung an Nur77 LBD und Nor1 LBD. Wenn Protein A-gereinigten Antikörper wurden Nurr1 weiter gegen Nur77 LBD und Nor1 LBD endgültigen affinitätsgerein...

Watch this Scientific Journal Video about Production of Nurr-1 Specific Polyclonal Antibodies Free of Cross-reactivity Against Its Close Homologs, Nor1 and Nur77 at JoVE.com

Production of Nurr-1 Specific Polyclonal Antibodies Free of Cross-reactivity Against Its Close Homologs, Nor1 and Nur77

Here, we selectively target antibodies against a specific member of a highly conserved family of proteins by immunizing animals with their most divergent regions followed by removing cross reactive antibodies by pre-adsorption.

Produktion von Nurr-1 spezifischen polyklonalen Antikörper Frei von Kreuzreaktivität gegen seine Close Homologe, Nor1 und Nur77

The nuclear receptor subfamily 4 (NR4A) is composed of 3 related proteins sharing a DNA binding domain (DBD) and a ligand-binding domain (LBD). The ...

production-nurr-1-specific-polyclonal-antibodies-free-cross

Research

JoVE Journal

Biology

7.4K Aufrufe.  McLean Hospital. Here, we selectively target antibodies against a specific member of a highly conserved family of proteins by immunizing animals with their most divergent regions followed by removing cross reactive antibodies by pre-adsorption.

Serie: Production of Nurr-1 Specific Polyclonal Antibodies Free of Cross-reactivity Against Its Close Homologs, Nor1 and Nur77

Principles of Site-Specific Recombinase (SSR) Technology

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular biology. Site-specific recombinases are proteins that bind to distinct DNA target sequences. The Cre/lox system was first described in bacteriophages during the 1980's. Cre recombinase is a Type I topoisomerase that catalyzes site-specific recombination of DNA between two loxP (locus of X-over P1) sites. The Cre/lox system does not require any cofactors. LoxP sequences contain distinct binding sites for Cre recombinases that surround a directional core sequence where recombination and rearrangement takes place. When cells contain loxP sites and express the Cre recombinase, a recombination event occurs. Double-stranded DNA is cut at both loxP sites by the Cre recombinase, rearranged, and ligated ("scissors and glue"). Products of the recombination event depend on the relative orientation of the asymmetric sequences.
SSR technology is frequently used as a tool to explore gene function. Here the gene of interest is flanked with Cre target sites loxP ("floxed"). Animals are then crossed with animals expressing the Cre recombinase under the control of a tissue-specific promoter. In tissues that express the Cre recombinase it binds to target sequences and excises the floxed gene. Controlled gene deletion allows the investigation of gene function in specific tissues and at distinct time points. Analysis of gene function employing SSR technology --- conditional mutagenesis -- has significant advantages over traditional knock-outs where gene deletion is frequently lethal.

Principles of Site-Specific Recombinase SSR Technology

The advent of site-specific recombinase (SSR) technology and the Cre/lox system has led to numerous advances in molecular biology, and has proven itself as a valuable tool for assessing gene function in transgenic animals. This interview discusses the mechanism of site specific recombination by Cyclization recombinase (Cre) and how the use of this enzyme has led to the development of conditional mutagenesis, which has significant advantages over traditional knock out strategies.

Principles of ortsspezifische Rekombinase (SSR)-Technologie

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular ...

Forschung

Biologie

Operant Learning of Drosophila at the Torque Meter

For experiments at the torque meter, flies are kept on standard fly medium at 25&deg;C and 60% humidity with a 12hr light/12hr dark regime. A standardized breeding regime assures proper larval density and age-matched cohorts. Cold-anesthetized flies are glued with head and thorax to a triangle-shaped hook the day before the experiment. Attached to the torque meter via a clamp, the fly's intended flight maneuvers are measured as the angular momentum around its vertical body axis. The fly is placed in the center of a cylindrical panorama to accomplish stationary flight. An analog to digital converter card feeds the yaw torque signal into a computer which stores the trace for later analysis. The computer also controls a variety of stimuli which can be brought under the fly's control by closing the feedback loop between these stimuli and the yaw torque trace. Punishment is achieved by applying heat from an adjustable infrared laser.

Measuring the yaw torque of tethered Drosophila with the torque meter allows the neuroscientist exquisite control of the stimulus situation of the experimental animal. Together with the unique genetic tools available in the fruit fly, this paradigm is used for a wide variety of neurobiological research.

Operanten Lernens von Drosophila an der Torque Meter

For experiments at the torque meter, flies are kept on standard fly medium at 25&deg;C and 60% humidity with a 12hr light/12hr dark regime. A standardized ...

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to measure odor evoked activity in the antennal lobe1. From then on, it has been continuously refined and applied to different neuropiles in the bee brain. Here, we describe the preparation currently used in the lab to measure activity in mushroom body neurons using a dextran coupled calcium-sensitive dye (Fura-2). We retrogradely stain mushroom body neurons by injecting dye into their axons or soma region. We focus on reducing the invasiveness, to achieve a preparation in which it is still possible to train the bee using PER conditioning. We are able to monitor and quantify the behavioral response by recording electro-myograms from the muscle which controls the PER (M17)2.
After the physiological experiment the imaged structures are investigated in greater detail using confocal scanning microscopy to address the identity of the neurons.

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

Bees can be conditioned in an appetitive olfactory learning paradigm (PER-conditioning). Using odors as stimuli, we established a method in which behavior is recorded while simultaneously Calcium Imaging is used to measure odor evoked activity in mushroom body neurons in vivo.

In vivo Ca 2 + - Imaging von Pilzkörper Neuronen während olfaktorischen Lernen in der Honigbiene

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

Autonom Bioluminescent Säugerzellen für die kontinuierliche und Echtzeit-Überwachung von Zytotoxizität

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

Design und Analyse des Temperaturpräferenzverhaltens und seines circadianen Rhythmus in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

Der Nachweis von intrazellulären Genexpression in lebenden Zellen der Maus, Mensch und Schwein Origin Mit Fluoreszenz-markierter Nanopartikel

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

Rückstands spezifische Einbau nichtkanonischer Aminosäuren in Modellproteine ​​eine Verwendung<em&gt; Escherichia coli</em&gt; Zellfreie Transkription-Translation-System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes ...

Use of Anti-phospho-girdin Antibodies to Visualize Intestinal Tuft Cells in Free-Floating Mouse Jejunum Cryosections

The actin binding protein girdin is a cytosolic protein that is required for actin remodeling to trigger cell migration in various tissues. Girdin is phosphorylated by both receptor and non-receptor tyrosine kinases at tyrosine 1798. Omori et al. developed site- and phosphorylation status-specific antibodies against human girdin at tyrosine-1798 (pY1798), which specifically bind to phosphorylated tyrosine-1798, but not to unphosphorylated tyrosine-1798. pY1798 antibodies have been used to specifically label tuft cells (TCs) that are present in mammalian gastrointestinal tissues, but the function of these cells is unclear. This protocol allows the robust visualization of TCs in the jejunum using pY1798 antibodies and immunofluorescence. To ensure successful and simple TC visualization, this protocol includes two histological techniques: production of free-floating cryosections from gelatin-filled jejunum tissue, and low-temperature antigen retrieval at 50 &#176;C for 3 h. Filling the jejunum with gelatin maintains the shape of free-floating sections throughout the staining procedure, whereas low-temperature antigen retrieval ensures robust signals from TCs. Successful use of this protocol results in pY1798 staining of TCs distributed from villus tip to crypt. Stained TCs have a spool-shaped soma and fluorescent signals condense at the lumenal tip, which corresponds to the protruding &#39;tuft.&#39;&#160;Phalloidin staining colocalized with pY1798-positive TCs at the thickened brush border, and corresponds to a rootlet mass extending from the TC tuft. This protocol could be used to examine TCs in human biopsy samples collected with gastrointestinal endoscopes. Furthermore, TCs were recently reported to accumulate following parasite infection in mice, suggesting that this protocol could have applications for diagnosis of parasite infections in the human gut.

Kuga et al. discovered that phosphorylation-status specific antibodies against the actin binding protein girdin phosphorylated at tyrosine 1798 (pY1798) can be used to label tuft cells (TCs). This protocol allows robust visualization of TCs using immunofluorescent staining of free-floating jejunum cryosections with pY1798 antibodies.

Verwendung von Anti-Phospho-girdin Antikörper gegen Darm Büschel Zellen frei schwebenden Maus Jejunum Cryosections visualisieren

The actin binding protein girdin is a cytosolic protein that is required for actin remodeling to trigger cell migration in various tissues. Girdin is ...

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2&#8211;3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

This work describes the use of CRISPR-Cas9 genome editing technology to knockout endogenous gene OsABCG15 followed by a modified Agrobacterium-mediated transformation protocol to produce a stable male-sterile line in rice.

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive ...

A Simple Dry Sectioning Method for Obtaining Whole-Seed-Sized Resin Section and Its Applications

The morphology, size and quantity of cells, starch granules&#160;and protein bodies in seed determine the weight and quality of seed. They are significantly different among different regions of seed. In order to view the morphologies of cells, starch granules and protein bodies clearly, and quantitatively analyze their morphology parameters accurately, the whole-seed-sized section is needed. Though the whole-seed-sized paraffin section can investigate the accumulation of storage materials in seeds, it is very difficult to quantitatively analyze the morphology parameters of cells and storage materials due to the low resolution of the thick section. The thin resin section has high resolution, but the routine resin sectioning method is not suitable to prepare the whole-seed-sized section of mature seeds with a large volume and high starch content. In this study, we present a simple dry sectioning method for preparing the whole-seed-sized resin section. The technique can prepare the cross and longitudinal whole-seed-sized sections of developing, mature, germinated, and cooked seeds embedded in LR White resin, even for large seeds with high starch content. The whole-seed-sized section can be stained with fluorescent brightener 28, iodine, and Coomassie brilliant blue R250 to specifically exhibit the morphology of cells, starch granules, and protein bodies clearly, respectively. The image obtained can also be analyzed quantitatively to show the morphology parameters of cells, starch granules, and protein bodies in different regions of seed.

This technique allows for the fast and simple preparation of whole-seed-sized resin section for the observation and analysis of cells, starch granules, and protein bodies in different regions of the seed.