We thank Ueli Grossniklaus (University of Z&#252;rich) for technical and financial support. We are thankful to Valeria Gagliardini, Christof Eichenberger, Arturo Bolanos and Peter Kopf for general lab support. This research was funded by the University of Z&#252;rich, grants from the Swiss National Foundation to CB (31003A_130722) and Ueli Grossniklaus (31003A_141245 and 31003AB-126006), and the Agence Nationale de la Recherche to DG (Programme ANR-BLANC-2012).

La procédure est décrite dans le flux de travail dans la figure 1, et la configuration de la dissection et l&#39;incorporation de tissus sont présentés dans la figure 2. 1. Fixation tissulaire <ol><li> Recueillir 20-30 carpelles dans un tube de centrifugeuse contenant fraîchement tampon fixateur BVO sur la glace. </li><li> Fixer le tissu 30 min en agitant doucement à température ambiante. </li><li> Faites tourner les tubes contenant les carpelles en fixateur dans une paillasse centrifuger 1 min à 400 x g. </li><li> Retirez soigneusement le tampon fixateur et ajouter 1 ml de PBT, placer les tubes sur la glace. </li></ol> 2. Dissection and Embedding <ol><li> Préparer cinq tubes Eppendorf avec chacun 200 pl d&#39;une fraîchement préparé, 5% d&#39;acrylamide mélange. </li><li> Préparer cinq lames Superfrost pré-nettoyées à l&#39;éthanol à 70% et étiquetés avec un crayon. </li><li> Décongeler une aliquote de 20% et 20% APS NaPS chacun, sur la glace. </li><li> Prenez 4-5 carpelles avec une pointe coupe-fin, le lieueux sur une lame propre, éliminer l&#39;excès de liquide. </li><li> Faire des coupes longitudinales avec une aiguille fine et détacher les murs de carpelles de libérer les lignes de ovules comme le montre la figure 2, éviter le séchage en couvrant avec du PBS (pas plus de 10 pi). </li><li> Ajouter rapidement et mélanger 12 NaPS pi, 12 pi APS avec une aliquote de 200 ul acrylamide mélange. </li><li> Ajouter 30 ul de l&#39;acrylamide activé sur les ovules disséqués. </li><li> Couvrir avec une lamelle mm 20 x 20, laisser polymériser à température ambiante, 45-60 min. </li><li> Retirer la lamelle couvre-objet en utilisant une lame de rasoir. A ce stade, les échantillons peuvent être conservés pendant une nuit à 4 ° C dans une jarre de Coplin contenant du PBS. </li></ol> 3. Traitement des tissus NOTE: Toutes les étapes, sauf 3.2.1, 3.2.3, 3.3.2 et 3.4.3 sont effectués dans des bocaux de Coplin avec 80 ml de solution sous le capot chimique à température ambiante. Les lames sont transférés avec une pointe plate-pince. <ol><li> clarification des tissus etfixation: <ol><li> Incuber 5 min dans du methanol. </li><li> Incuber 5 min dans de l&#39;éthanol. </li><li> Incuber 30 min dans de l&#39;éthanol: xylène (1:1). </li><li> Incuber 5 min dans de l&#39;éthanol. </li><li> Incuber 5 min dans du methanol. </li><li> Incuber 15 min dans du methanol et le PBT (1:01), complété avec 2,5% de formaldehyde. </li><li> Rincer 2 x 10 min en PBT. A ce stade, les lames peuvent être conservées pendant une nuit à 4 ° C. </li></ol></li><li> paroi cellulaire digestion: <ol><li> Décongeler une aliquote de la paroi cellulaire digestion mélange sur de la glace. </li><li> Prenez une diapositive de la cuve à rainures, drainer l&#39;excès de liquide en le plaçant à la verticale sur une serviette en papier. </li><li> Ajouter 100 pi de la paroi cellulaire digestion mélange sur le pavé de l&#39;acrylamide et couvrir avec une lamelle 23 mm x 46. Répétez l&#39;opération pour les autres diapositives. Incuber pendant 2 heures à 37 ° C dans une chambre humide (décrit dans Matériels). </li><li> Laver les lames 2 x 5 min en PBT. </li></ol></li><li> RNase A de traitement: <ol><li> Prenez une diapositive de la cuve à rainures, égoutter èmee excès de liquide que précédemment. </li><li> Incuber chaque lame avec 100 pl de RNase A à 100 ug / ml dans du PBS avec 1% de Tween-20 pendant 1 h à 37 ° C dans une chambre humide. </li><li> Laver les lames de 2 x 5 min en PBT. </li></ol></li><li> Post-fixation et perméabilisation: <ol><li> Post-fixer pendant 20 min dans fraîchement PBT-F. </li><li> Rincer les lames pendant 10 minutes dans le PBT. </li><li> Perméabiliser pendant 2 h dans du PBS avec 2% de Tween-20 à 4 ° C. </li><li> Rincer les lames de 2 x 5 min en PBT. </li></ol></li></ol> 4. Immunocoloration NOTE: Pour cette étape, la concentration optimale de l&#39;anticorps primaire doit être testée en utilisant différentes dilutions (1:200, 1:500, 1:1000) d&#39;anticorps. <ol><li> Incuber chaque lame avec 100 pi d&#39;anticorps primaire dilué dans du PBS avec 0,2% de Tween-20 pendant 12 à 24 heures à 4 ° C. </li><li> Laver les lames en PBT pour 2-4 heures à température ambiante sous agitation douce. </li><li> Appliquer le1:200 d&#39;anticorps secondaire dans du PBS + 0,2% de Tween-20 pendant 24 h à 4 ° C. </li><li> Laver les lames dans PBT pendant 1 heure à température ambiante sous agitation douce. </li><li> Contre-colorer avec 10 pg / ml d&#39;iodure de propidium dans du PBS pendant 15 min, puis rincer 15 min dans du PBS sous agitation douce, à température ambiante. </li><li> Mont en milieu de montage anti-décoloration de liquide supplémenté avec 10 pg / ml d&#39;iodure de propidium. Que le milieu de montage durcir pendant 1 heure avant l&#39;acquisition d&#39;images par CLSM. </li></ol> 5. Imagerie quantitative <ol><li> acquisition de l&#39;image: <ol><li> Acquérir des images de haute résolution en utilisant CLSM, de préférence en utilisant un mode de balayage de résonance, ce qui permet une meilleure conservation des signaux de fluorescence au cours de l&#39;imagerie 23 prolongée, et une lentille à immersion 63X de glycérol. </li><li> Testez les paramètres d&#39;acquisition tels que l&#39;intensité du laser, le gain, sténopé, la taille de voxel et le facteur de zoom au début de l&#39;expérience de définir une procédure d&#39;acquisition standard pour suivre strictement tout au long detoutes les diapositives pour des mesures quantitatives cohérentes. </li><li> Vérifier l&#39;absence de diaphonie entre fluorochromes. Le cas échéant, mettre en place une analyse séquentielle. Acquérir les images de transmission séparément et non simultanément. </li><li> Effectuer une acquisition d&#39;image de série, en trois dimensions avec la plus haute résolution possible dans les dimensions X et Y et avec 2x suréchantillonnage dans la dimension z (la règle de Nyquist). </li></ol></li><li> Traitement de l&#39;image: <ol><li> Reconstruire des images de série en trois dimensions en utilisant un logiciel de source commerciale ou ouvert. </li><li> Définir des surfaces de contour autour de chaque noyau (ou cellule) d&#39;intérêt en 3D. </li><li> Quantifier la fluorescence dans chaque canal à la somme des intensités de pixels dans chaque objet. </li><li> Exporter les données vers Excel pour des analyses statistiques. Normaliser les signaux d&#39;anticorps contre les signaux de coloration par exemple d&#39;ADN. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Chez les plantes à fleurs, la lignée reproducteur féminin est entouré de plusieurs couches de cellules, y compris le nucelle et les téguments de l&#39;ovule, ce qui rend la coloration cytologique en tout montage techniquement difficile. Nous présentons ici un protocole efficace permettant à l&#39;élaboration et au traitement d&#39;un grand nombre d&#39;ovules appropriés pour la coloration cytologique comme immunologique, la coloration de l&#39;ADN et l&#39;hybridation fluorescente in situ l&#39;ensembl...

Chez les plantes à fleurs, l&#39;établissement de lignées de reproduction commence par la différenciation des CML, MMC féminine et masculine cellule microspores mère. La MMC se développe à partir d&#39;une cellule nucellaire sous-épidermique à l&#39;extrémité distale de l&#39;ébauche ovule, et la cellule mère des microspores se développe à partir du tissu sporogène dans la loge des anthères, qui sont situés profondément à l&#39;intérieur des organes floraux 1. SMC subissent une méiose pour produire des spores haploïdes, qui donnent alors lieu à des gamétophytes sur la mitose. Le gamétophyte femelle, ou sac embryonnaire, se compose d&#39;un ovule, une cellule centrale, deux synergides et trois antipodes. Le gamétophyte mâle, ou le pollen, est composé d&#39;une cellule végétative et deux spermatozoïdes. Alors que le gamétophyte mâle reste une étude d&#39;objet de relativement accessible, le gamétophyte femelle est incorporé dans l&#39;ovule, se joint à la carpelle de fleur, et pose donc des défis particuliers pour les analyses moléculaires et cytologiques. Récemment, cependant, assistée par lasermicrodissection offert une solution élégante permettant des analyses du transcriptome dans la console MMC et les cellules gamétophytiques femmes 2-4. En plus de l&#39;expression du gène candidat analyses, par exemple, en utilisant l&#39;hybridation in situ de l&#39;ARN ou des dosages de gène rapporteur dans les analyses cytologiques permet l&#39;étude des dynamiques de composants cellulaires endogènes en utilisant une coloration spécifique cellulaire directe ou indirecte immunomarquage. En particulier, la coloration cytogénétique utilisant FISH et coloration de l&#39;ADN, avec immunomarquage des modifications de la chromatine ou des composants de la chromatine sont des approches centrales à élucider la dynamique de la chromatine et l&#39;organisation nucléaire chez Arabidopsis 5. Typiquement, la méiose entraîne la dynamique des chromosomes spécifiques qui a été bien étudiés dans plante mâle méiocytes 6,7; encore à grande échelle, la réorganisation de la chromatine spécifique de la cellule, ce qui reflète probablement la reprogrammation épigénétique dynamique a été décrite au cours du développement du pollen 8-10. En revanche, en raisonà l&#39;inaccessibilité relative de la meiocyte femelle et gamétophyte, ces enquêtes restent techniquement difficile à appliquer, et nécessitent souvent dissection sectionnement ou manuel et la digestion enzymatique (voir ci-dessous). En outre, l&#39;absence fréquente de clarté optique dans l&#39;ensemble du montage est un obstacle à l&#39;imagerie haute résolution des cellules reproductrices dans ovules intacts. Une méthode classique pour l&#39;analyse cytologique de l&#39;organisation des chromosomes entiers dans des ovules de montage utilise la coloration de Feulgen 11-13. Elle implique une hydrolyse acide (en utilisant de l&#39;acide hypochloreux) de l&#39;ADN qui en résulte provoque la dénaturation des protéines et la destruction de la structure de la chromatine ainsi. Alternativement, l&#39;organisation des chromosomes dans méiocytes femmes et les cellules gamétophytiques peut être observée en utilisant la coloration DAPI et immunomarquage sur coupes semi-fines ou sacs embryonnaires disséqués et MMC (par exemple voir 14-18). Il est clair, cependant, la dissection manuelle et la coupe peut être beaucoup de travail etentrave sur l&#39;analyse qualitative et quantitative d&#39;un grand nombre d&#39;épitopes de la chromatine. Ici, nous fournissons un protocole efficace pour préparer un grand nombre d&#39;ovules Arabidopsis pour une variété de coloration cytologique en aval dans l&#39;ensemble du montage. En bref, les boutons floraux sont incubées dans une solution de fixation, des rangées de ovules sont disséqués du carpelle et intégrés dans l&#39;acrylamide sur la diapositive que fait pour méiocytes de pollen 19,20. Les ovules sont incorporés en outre compensées et fixés dans le méthanol, l&#39;éthanol, le xylène et avant la digestion de la paroi cellulaire et de perméabilisation. Des variations possibles de ces étapes sont décrites. Les échantillons peuvent ensuite être utilisés pour la coloration de l&#39;ADN, une immunocoloration et FISH. Le mode de préparation est efficace et permet parallèle expérimental (jusqu&#39;à 16 lames peuvent être préparés d&#39;une journée d&#39;analyse en aval différent). Les traitements décrits permettent signaux homogènes dans l&#39;ensemble du montage et bien conservés histologique, cellulaire,et l&#39;organisation nucléaire dans les cellules reproductrices et les cellules environnantes qui bénéficient nucellaires comparaisons qualitatives et quantitatives entre les types cellulaires. Calibré, l&#39;imagerie à haute résolution basée sur CLSM suivie d&#39;une reconstruction en 3 dimensions permet des mesures quantitatives significatives de signaux fluorescents. Nous avons utilisé avec succès cette procédure pour analyser la dynamique de la chromatine dans la console MMC de différenciation 21 et le développement gamétophyte femelle 22; Nous présentons ici des résultats représentatifs de l&#39;analyse de l&#39;hétérochromatine, immunologique de la chromatine, la GFP immunologique et FISH dans l&#39;ensemble du montage ovules. Nous croyons en outre que notre protocole sera adapté pour d&#39;autres tissus végétaux et des espèces.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;"><a name="RANGE!A1:D39">Name of Material/ Equipment</a></td>
			<td style="width:135px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:481px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Solutions</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">BVO Fixation Buffer (based on32)</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">2mM EGTA, pH7.5, 1% (v/v) Formaldehyde,10% DMSO, 1&#215; PBS, 0.1% Tween-20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBT</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">1&#215; PBS, 0.1% Tween-20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBT-F</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">1&#215; PBT, 2.5% (v/v) formaldehyde</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">30% acrylamide:bisacrylamide&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">3g acrylamide, 0.33g bisacrylamide, 1&#215; PBS (prepare 10ml, store at 4&#176;C)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">200mL&#160; 5% acrylamide mix in PBS&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">34mL 30% acrylamide:bisacrylamide, 166mL 1&#215; PBS (make fresh from 30% stock)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">20% ammoniumpersulfte&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">0.2g ammoniumpersulfte, 1mL sterile water (prepare aliquots with 1mL and store at -20&#176;C)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">20% sodium sulfite&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">0.2g sodium sulfite, 1mL sterile water (prepare aliquots with 1mL and store at -20&#176;C)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell wall enzyme mix</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">0.5% (w/v) cellulase, 1% (w/v) driselase, 0.5% (w/v) pectolyase</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Reagents and Materials</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F1635</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma</td>
			<td>D5879</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris</td>
			<td>Amaresco</td>
			<td>0497</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Schaurlau</td>
			<td>ET00102500</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>Schaurlau</td>
			<td>ME03062500</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylene</td>
			<td>ROTH</td>
			<td>4436.1</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellulase</td>
			<td>Sigma</td>
			<td>1794</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Driselase</td>
			<td>Sigma</td>
			<td>D8037</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pectolyase</td>
			<td>Sigma</td>
			<td>P5936</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Merck</td>
			<td>8.22184.0500</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA</td>
			<td>Sigma</td>
			<td>E-4378</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">acrylamide</td>
			<td>Sigma</td>
			<td>A-3553</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bisacrylamide</td>
			<td>Sigma</td>
			<td>M2022</td>
			<td style="width:481px;">toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ammoniumpersulfate</td>
			<td>Sigma</td>
			<td>A9164</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium sulfite</td>
			<td>Fluka</td>
			<td>71988</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-trimethyl-Histone H3 (Lys4)</td>
			<td>Upstate</td>
			<td>07-473</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti- monomethyl-Histone H3 (Lys27)</td>
			<td>Upstate</td>
			<td>07-448</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488~goat ~anti ~rabbit (H+L)</td>
			<td>Molecular Probe</td>
			<td>A11008</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProlongGold</td>
			<td>Invitrogen</td>
			<td>P36934</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propidium iodide</td>
			<td>Sigma</td>
			<td>P4170</td>
			<td style="width:481px;">toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td style="width:481px;">toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNAse A</td>
			<td>Roche</td>
			<td>10109169001</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coplin jar</td>
			<td>Huber &#38; CO</td>
			<td>10.055</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps</td>
			<td>DUMONT BIOLOGY</td>
			<td style="width:119px;"></td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shaker</td>
			<td>Heidolph</td>
			<td>543-12310-00-0</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Moist chamber</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">A plastic box with damp paper towel inside, a plastic support is put into the box for supporting the slides and keep slides from the water.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfrost Plus slide</td>
			<td>Thermo Fisher</td>
			<td>J1800AMNZ</td>
			<td style="width:481px;">Menzel-Gl&#228;ser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FISH Tag DNA KIt</td>
			<td>Invitrogen</td>
			<td>F32947</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">GFP booster</td>
			<td style="width:135px;">Chromotek</td>
			<td style="width:119px;"></td>
			<td style="width:481px;"></td>
		</tr>
	</tbody>
</table>

an efficient method for quantitative, single-cell analysis of chromatin modification and nuclear architecture in whole-mount ovules in arabidopsis

Chez les plantes à fleurs, la transition du destin cellulaire somatique à la reproduction est marquée par la spécification des cellules mères de spores (SMC) dans les organes floraux de la plante adulte. Le SMC femelle (cellule mère de mégaspore, MMC) distingue dans l&#39;ébauche ovule et subit la méiose. Le mégaspore haploïde sélectionné est ensuite soumis à la mitose pour former le gamétophyte femelle multicellulaire, qui donnera naissance aux gamètes, l&#39;ovule et la cellule centrale, avec des cellules accessoires. L&#39;accessibilité limitée de la console MMC, meiocyte et gamétophyte femelle à l&#39;intérieur de l&#39;ovule est techniquement difficile pour cytologique et cytogénétique des analyses au niveau d&#39;une seule cellule. En particulier, immunodétection directe ou indirecte d&#39;épitopes cellulaires ou nucléaires est affaiblie par la faible pénétration des réactifs à l&#39;intérieur de l&#39;imagerie cellulaire végétale et une seule cellule est affermé par le manque de clarté optique dans l&#39;ensemble du montage tissus. Ainsi, nous avons développé une méthode efficace pour analyser les nucll&#39;organisation de l&#39;oreille et la modification de la chromatine à haute résolution de la cellule unique dans l&#39;ensemble du montage ovules d&#39;Arabidopsis embarqués. Il est basé sur la dissection et l&#39;incorporation d&#39;ovules fixes dans une fine couche de gel d&#39;acrylamide sur une lame de microscope. Les ovules embarqués sont soumis à des traitements chimiques et enzymatiques visant à améliorer la clarté des tissus et de la perméabilité des réactifs d&#39;immunomarquage. Ces traitements préserver organisation, ADN et de protéines épitopes cellulaires et la chromatine. Les échantillons peuvent être utilisés pour différentes analyses cytologiques aval, y compris la chromatine immunocoloration, l&#39;hybridation in situ par fluorescence (FISH), et la coloration de l&#39;ADN pour l&#39;analyse de l&#39;hétérochromatine. La microscopie confocale à balayage laser (CLSM), l&#39;imagerie à haute résolution, suivie d&#39;une reconstruction 3D permet des mesures quantitatives à seule cellule de résolution.

Nous fournissons un protocole robuste pour la préparation à grande échelle et le traitement des ovules Arabidopsis appropriés pour la coloration cytologique dans l&#39;ensemble du montage. Merci à l&#39;encastrement, les ovules conservent une structure en 3 dimensions (Figure 3). En outre, le traitement des tissus, y compris la clarification optique permet des structures sous-cellulaires d&#39;imagerie à haute résolution. Figure 4 montre coloration de l&#39;ADN dans l&#3...

Watch this Scientific Journal Video about An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis at JoVE.com

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

Nous fournissons ici un protocole efficace et fiable pour immunologique, hybridation fluorescente in situ, la coloration de l&#39;ADN suivie quantitative, imagerie à haute résolution dans des ovules d&#39;Arabidopsis thaliana l&#39;ensemble du montage. Cette méthode a été utilisée avec succès pour analyser des modifications de la chromatine nucléaire et de l&#39;architecture.

Une méthode efficace pour quantitative, analyse unicellulaire de la chromatine Modification et architecture nucléaire dans l&#39;ensemble du montage ovules dans<em&gt; Arabidopsis</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

an-efficient-method-for-quantitative-single-cell-analysis-chromatin

Research

JoVE Journal

Biology

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

13.1K vues.  University of Zürich.  Nous fournissons ici un protocole efficace et fiable pour immunologique, hybridation fluorescente in situ, la coloration de l'ADN suivie quantitative, imagerie à haute résolution dans des ovules d'Arabidopsis thaliana l'ensemble du montage. Cette méthode a été utilisée avec succès pour analyser des modifications de la chromatine nucléaire et de l'architecture. 

Vidéo: An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Double Mont entier hybridation in situ d&#39;embryons de poulet début

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

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Biologie

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Méthode de coloration Mont anticorps entier de Chick

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Salut-C: une méthode d&#39;étude de l&#39;architecture tridimensionnelle des génomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualisation et d&#39;analyse de molécules d&#39;ARNm par fluorescence<em&gt; In Situ</em&gt; Hybridation dans<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation.

The material here describes a method developed to preserve the three-dimensional chromatin structure of testicular germ cells. This has been termed the three-dimensional (3D) slide method. This method improves sensitivity for detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH).

Procédé de préparation de lame pour préserver l’architecture de chromatine tridimensionnelle de cellules germinales testiculaires

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Mesure spatiale et temporelle Ca<sup&gt; 2 +</sup&gt; Signaux de plantes d&#39;Arabidopsis

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a &#39;whole mount&#39; tissue preparation, or developmental stage (such as an embryo or larva) of interest. WMISH is extremely powerful because it can significantly contribute to the functional characterization of complex metazoan genomes, a challenge that is becoming more of a bottleneck with the deluge of next generation sequence data. Despite the conceptual simplicity of the technique much time is often needed to optimize the various parameters inherent to WMISH experiments for novel model systems; subtle differences in the cellular and biochemical properties between tissue types and developmental stages mean that a single WMISH method may not be appropriate for all situations. We have developed a set of WMISH methods for the re-emerging gastropod model Lymnaea stagnalis that generate consistent and clear WMISH signals for a range of genes, and across all developmental stages. These methods include the assignment of larvae of unknown chronological age to an ontogenetic window, the efficient removal of embryos and larvae from their egg capsules, the application of an appropriate Proteinase-K treatment for each ontogenetic window, and hybridization, post-hybridization and immunodetection steps. These methods provide a foundation from which the resulting signal for a given RNA transcript can be further refined with probe specific adjustments (primarily probe concentration and hybridization temperature).

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.

A Mont entier<em&gt; In Situ</em&gt; Méthode Hybridation pour le mollusque gastéropode<em&gt; Lymnaea stagnalis</em

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

Protocole CUBIC Visualise Protein Expression à cellule unique Résolution dans les préparations entières de la peau de montage

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

Visualisation de la Structure du tissu adipeux blanc 3D utilisant entier-Montez la souillure

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in turn requires accurate determination of disease phenotypes of plants upon infection with a particular fungal pathogen. However, accurate disease phenotyping with unculturable biotrophic fungal pathogens such as powdery mildew is not easy to achieve and can be a rate-limiting step of a research project. Here, we have developed a safe, efficient, and easy-to-operate disease phenotyping system using the Arabidopsis-powdery mildew interaction as an example. This system mainly consists of three components: (i) a wooden inoculation box fitted with a removable lid mounted with a stainless steel or nylon mesh of ~50 &#181;m pores for inoculating a flat of plants with fungal spores, (ii) a transparent plastic chamber with a small front opening for minimizing spore escape while conducting inoculation inside, and (iii) a spore-dislodging and distribution method for even and effective inoculation. The protocols described here include the steps and parameters for making the inoculation box and the plastic chamber at a low cost, and a video demonstration of how to use the system to enable even inoculation with powdery mildew spores, thereby improving accuracy and reproducibility of disease phenotyping.

We present a protocol for constructing a simple spore-distribution system consisting of an inoculation box with a ~50 &#181;m mesh and a transparent plastic chamber. This can be used to evenly inoculate plants with powdery mildew spores, thereby enabling accurate and reproducible assessment of disease phenotypes of plants under study.

Une méthode d’inoculation simple et flexible pour évaluer avec précision les phénotypes d’oïdium d’Arabidopsis et d’autres plantes

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in ...