Ce travail a été soutenu par les US National Institutes of Health accorde HL082798 et HL111580.

Une. coupes de tissus rénaux <ol><li> Fixés au formol (10%) des sections de rein paraffine sont coupés sur des lames de microscope à 5 mm d&#39;épaisseur en utilisant un Microm HM 355 S. Les lames peuvent être stockées pendant plusieurs mois avant l&#39;analyse. </li></ol> 2. Préparation de la solution <ol><li> Préparer 1 L de PBS 1x dans le trou DDH 2 O dans une bouteille avec vis autoclavable. Remplir une autre bouteille de 1 L de ddH 2 O. Ajouter 1 ml de DEPC à chaque bouteille, couvercle et agiter vigoureusement. Que les solutions reposer toute la nuit à température ambiante pour détruire RNAses puis autoclave. Ajouter 400 ml de Tween-20 à 1 L de PBS 1x à faire du PBS-T. </li><li> Préparer le tampon de la protéinase K (50 mMTris-HCl, pH 8,0 et 1 mM de CaCl2 dans de l&#39;eau traitée au DEPC). </li><li> Préparer une solution de 0,2% de glycine dans du PBS-T. </li></ol> 3. Préparation des tissus <ol><li> Préparer un grand volume de tampon d&#39;hybridation (de 50 à 100 ml), composée de 50% de formamide, 5x SSC, 0,1% de Tween, 9,2mM d&#39;acide citrique pour ajuster le pH à 6, 50 pg / ml d&#39;héparine, et 500 pg / ml d&#39;ARN de levure dans DEPC traitées ddH 2 O. Diviser en aliquotes de 1 ml et conserver à -80 ° C. </li><li> Retirez la paraffine du tissu par lavage 3x dans le xylène frais pendant 5 minutes chacun. </li><li> Réhydrater les coupes de tissu par incubation 5 min dans des concentrations décroissantes d&#39;éthanol (100%, 75%, 50% et 25%) dans le trou DDH 2 O. </li><li> Traiter les diapositives avec DEPC suffisamment pour couvrir complètement les coupes de tissu (environ 0,4 à 0,5 ml) pendant 1 min. Assurez-vous que les tissus ne se dessèchent pas. </li><li> Rincer les lames dans DEPC traitées PBS-T deux fois pendant 5 min, recueillir le DEPC contenant des déchets pour une élimination appropriée. Tous les réactifs doivent être DEPC traitées pour éliminer RNAses partir de ce point. </li><li> Préchauffer la protéinase K en mémoire tampon et ajouter la protéinase K à une concentration finale de 10 pg / ml. Couvrir les coupes de tissus avec une solution de protéinase K et incuber à 37 º C pendant 5 min. </li><li> Supprimer rapidement les proteinassolution e K et ajouter 0,2% de glycine dans du PBS-T pendant 30 sec. </li><li> Rincer les lames dans DEPC traitées PBS-T deux fois pendant 30 secondes chacun. </li><li> Fixer sections fraîchement paraformaldéhyde 4% pendant 10 min. </li></ol> 4. Hybridation <ol><li> Ajouter 50 ul de tampon d&#39;hybridation (formamide 50%, 5x SSC, 0,1% de Tween, 9,2 mM d&#39;acide citrique pour ajuster le pH à 6, 50 pg / ml d&#39;héparine, 500 ug / ml d&#39;ARN de levure) par coupe de tissu et recouvrir avec de la RNase HybriSlips libres couper juste plus grande que les sections de tissu. </li><li> Sections Prehybridize à humidité contrôlée four à hybridation pendant 2 heures à la température d&#39;hybridation calculée pour le «sonde digoxigénine marqué 3 (habituellement l&#39;ADN Tm de la sonde -21 ° C, (c&#39;est à dire 54 ° C pour miR-382, la sonde ADN Tm = 75 ° C ), en utilisant des lingettes de laboratoire de tissus trempés dans 50% formamide/50% 5x SSC à garder la chambre humidifié. </li><li> Diluer la sonde miARN, le contrôle positif (U6, petit ARN nucléaire) et de contrôle négatif (brouillésséquence) à 40 nM dans du tampon d&#39;hybridation (75 pi de tampon est nécessaire pour la section). </li><li> Chauffer la sonde dans le tampon d&#39;hybridation à 65 ° C pendant 5 min. </li><li> Retirer les lames de l&#39;hybridation four et retirer les lamelles et le tampon d&#39;hybridation excès de pré-hybridation. </li><li> Ajouter 75 ul de sonde contenant un tampon par section de tissu, directement au tissu. Couvrir tissu avec HybriSlips juste assez larges pour couvrir les coupes de tissus. </li><li> Garder le niveau de support de diapositives, de retour à l&#39;hybridation four pour une nuit d&#39;incubation à la température de l&#39;étape 4.2 (environ 16 heures). </li></ol> 5. Rigueur de lavage <ol><li> Ajouter 200 ul de 2 x SSC, on chauffe à la température d&#39;hybridation, un peu moins d&#39;un bord de la lamelle pour aider à libérer la lamelle couvre-objet à partir du tissu. Retirer la lamelle en inclinant la lame. </li><li> Laver les lames dans 200 ul de formamide à 50%, 50% SSC 2x à 3x hybridation de température pendant 30 minutes chacun. </li><li> Rincerles lames 5x dans du PBS-T à la température ambiante pendant 5 min chacun sur agitateur orbital à vitesse réduite. </li></ol> 6. Détection <ol><li> Incuber lame dans du tampon de blocage (2% de sérum de chèvre ou de cheval, 2 mg / ml de BSA dans du PBS-T) pendant 1 heure à température ambiante sur un agitateur orbital à vitesse réduite. </li><li> Diluer fragments anti-DIG-AP Fab dans un tampon de blocage à 1:100. Ajouter 100 ul par section de tissu et couvrir avec HybriSlips coupé juste assez grand pour couvrir la section. </li><li> Incuber l&#39;anticorps dans une chambre humidifiée à 4 ° C pendant une nuit (environ 16 heures). </li><li> Laver les lames dans du PBS-T 7x, pendant 5 min chacun sur agitateur orbital à vitesse réduite. </li><li> Laver les lames dans du tampon AP (100 mM Tris-HCl pH 9,5, 50 mM de MgCl2, 100 mM de NaCl, 0,1% Tween 20) 3x, pendant 5 minutes chacune. </li><li> Ajouter la solution de NBT / BCIP de tampon AP (200 μl/10 tampon ml) </li><li> Ajouter 400-500 ml de solution diluée NBT / BCIP à chaque diapositive pour couvrir complètement toutes les sections de tissus. Développer dans un endroit sombre, niveau, Humidified chambre pour 4-5 heures. </li></ol> 7. Diapositives de montage <ol><li> Déshydrater sections dans des concentrations croissantes d&#39;éthanol (25%, 50%, 75%, 100%) pendant 5 min chacun. </li><li> Incuber dans 5x xylène afin de dégager sections. </li><li> Mont glisse dans Permount milieu de montage et sécher pendant la nuit. </li></ol>

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Il n&#39;y a rien à divulguer.

Le but de cet article est de décrire un protocole de miARN hybridation in situ qui fonctionne bien dans les tissus rénaux fixés au formol. Tout en travaillant sur ce protocole plusieurs sources importantes d&#39;artefact coloration ont été identifiés. Une attention particulière à ces points peut aider à éviter la coloration artefact et augmenter la probabilité d&#39;une course de ISH succès. Une des causes les plus évitables de artefact coloration peut se produire lorsq...

MicroARN sont de petite taille (environ 22 nucléotides de long) non codantes des ARN qui sont produites de façon endogène. Ils fonctionnent généralement à supprimer l&#39;expression de la protéine par la répression de translation ou la dégradation de l&#39;ARNm. miARN se lient à des cibles ARNm avec complémentarité incomplète, ce qui permet à un seul miARN pour supprimer plusieurs cibles. Comprendre les types et les structures cellulaires expriment miARN est une partie importante de la compréhension des mécanismes par lesquels des modifications dans miRNA influence d&#39;expression cellulaire et des phénotypes de tissus. Bien que les méthodes telles que le séquençage miRNA, qPCR et Northern blot peuvent être utilisés pour la détection de miARN dans les tissus entiers, cette approche ne permet pas de déterminer quel type de cellule spécifique ils sont venus dans un tissu donné. Dissection de composants cellulaires et structurelles avant l&#39;analyse à l&#39;aide de ces méthodes peut être très difficile et les conditions nécessaires à la réalisation des isolations appropriées peut conduire à alterations dans l&#39;expression génique ou de la dégradation d&#39;ARN. microRNA hybridation in situ est une méthode utilisée pour visualiser l&#39;emplacement de micro-ARN et des niveaux d&#39;expression dans les tissus. Cette technique est particulièrement utile dans les tissus constitués de structures hétérogènes, tels que le rein. Les microARN ont été montré à jouer un rôle de régulation dans 2,3 de développement du rein et de la physiologie 4. modifications d&#39;expression de microARN ont également été montré pour être impliqué dans la pathologie rénale telles que la fibrose 5-10, la néphropathie diabétique 7, carcinome rénal 11,12, et l&#39;insuffisance rénale aiguë 13. Dans notre recherche, nous avons constaté que l&#39;optimisation de microARN hybridation in situ pour les tissus rénaux en était précieux pour déterminer les emplacements exacts structurelles d&#39;expression des miARN dans la santé et la maladie 14. Détermination de l&#39;expression tubulaire et cellulaire de différentes microARN est important parce que leur réglementation de targets peuvent dépendre de fonctions cellulaires. Dans les états pathologiques, il est également important de déterminer comment altérations de l&#39;expression des miARN peuvent avoir des répercussions sur la fonction. Le but de la méthode décrite ici était de construire des méthodologies ISH existants élaborés par Kloosterman et al. 15, d&#39;autres enquêteurs 16,17, et ceux suggérés par Exiqon 1 et d&#39;optimiser la méthode pour les tissus rénaux fixes au formol. Nous avons utilisé cette méthode avec succès pour identifier les différences régionales dans les microARN miR rénale expression-382 avec obstruction urétérale unilatérale 18. Cette approche peut être utilisée avec d&#39;autres tissus, avec une optimisation supplémentaire.

<table border="0" cellpadding="0" cellspacing="0" width="481">
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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Paraformaldehyde</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">P6148</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">PBS</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:137px;">70011044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Diethyl Pyrocarbonate</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">D5758</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Tween-20</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">P1379</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Xylenes</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">534056</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Ethanol</td>
			<td style="width:71px;">Ultra Pure</td>
			<td style="width:137px;">200CSPTP</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Tris-HCl</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:137px;">15506-017</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">CaCl2</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">C2536</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Glycine</td>
			<td style="width:71px;">J.T. Baker</td>
			<td style="width:137px;">4059-00</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Citric Acid</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">C2404</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Formamide</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">F9037</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">20x SSC Buffer</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:137px;">15557-044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Heparin</td>
			<td style="width:71px;">SAGENT</td>
			<td style="width:137px;">25021-400-30</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Yeast RNA</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:137px;">AM7118</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">MgCl2</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">M8266-1004</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">NaCl</td>
			<td style="width:71px;">J.T. Baker</td>
			<td style="width:137px;">4058-01</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Proteinase K</td>
			<td style="width:71px;">Fermentas</td>
			<td style="width:137px;">EO0491</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">3&#8217; DIG- miRNA probe</td>
			<td style="width:71px;">Exiqon</td>
			<td style="width:137px;">miR dependent-05</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">3&#8217; DIG- Scrambled miR probe</td>
			<td style="width:71px;">Exiqon</td>
			<td style="width:137px;">99004-05</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">3&#8217; DIG- U6 miR probe</td>
			<td style="width:71px;">Exiqon</td>
			<td style="width:137px;">99002-05</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Anti-Digoxigenin-Ab Fab</td>
			<td rowspan="2" style="width:71px;">Roche</td>
			<td rowspan="2" style="width:137px;">11093274910</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Fragments</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">NBT/BCIP</td>
			<td style="width:71px;">Roche</td>
			<td style="width:137px;">11681451001</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Permount</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:137px;">SP15-500</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Coverslips</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:137px;">Fit to tissue size</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Microtom HM 355 S</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:137px;">23-900-672</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">In-slide Out Hybridization Oven</td>
			<td style="width:71px;">Boekel</td>
			<td style="width:137px;">241000</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Aluminum Tray Assembly</td>
			<td style="width:71px;">Boekel</td>
			<td style="width:137px;">C2403973</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Stainless Steel Rack Insert</td>
			<td style="width:71px;">Boekel</td>
			<td style="width:137px;">C2403754</td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

microrna in situ hybridization for formalin fixed kidney tissues

Dans cet article, on décrit un procédé de détection colorimétrique de miARN dans le rein par hybridation in situ avec de la digoxigénine marqué dans les sondes de microARN. Ce protocole, développé à l&#39;origine par Kloosterman et ses collègues pour une large utilisation de sondes Exiqon miRNA 1, a été modifié pour surmonter les difficultés inhérentes à l&#39;analyse des miRNA dans les tissus rénaux. Il s&#39;agit notamment des questions telles que l&#39;identification de la structure et difficile à retirer la sonde résiduelle et anticorps. L&#39;utilisation de relativement mince, 5 mm d&#39;épaisseur, les coupes de tissus autorisés pour une visualisation claire des structures du rein, alors qu&#39;un signal de la sonde solide a été maintenu dans les cellules. En outre, la concentration de la sonde et des conditions d&#39;incubation ont été optimisés pour faciliter la visualisation de l&#39;expression de microARN avec faible bruit de fond et le signal non spécifique. Ici, le protocole optimisé est décrit, couvrant la collecte de tissu initial et de la préparation à travers le montage de lames à la fin de la procédure. Le compone de basents de ce protocole peuvent être modifiés pour l&#39;application à d&#39;autres tissus et des modèles de culture cellulaire.

Les zones d&#39;une section de tissu qui se dessécher pendant les hybridations, des incubations ou étapes de lavage généralement finissent par coloration plus sombre lors de l&#39;élaboration du NBT / BCIP. Figure 1 montre une partie d&#39;une section de rein, dans lequel le HybriSlip glissé hors du bord d&#39; le tissu, lui permettant de devenir partiellement déshydraté. Malgré la réhydratation et la couverture dans les étapes restantes, le signal dans la partie déshydraté est artificielle...

Watch this Scientific Journal Video about MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues at JoVE.com

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

Cet article décrit un protocole d&#39;hybridation in situ optimisé pour la détection colorimétrique de l&#39;expression de micro-ARN dans des sections de rein fixés au formol.

MicroRNA<em&gt; In situ</em&gt; Hybridation pour les tissus rénaux fixés au formol

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

microrna-in-situ-hybridization-for-formalin-fixed-kidney-tissues

Research

JoVE Journal

Biology

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

14.8K vues.  Medical College of Wisconsin.  Cet article décrit un protocole d'hybridation in situ optimisé pour la détection colorimétrique de l'expression de micro-ARN dans des sections de rein fixés au formol. 

Vidéo: MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

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

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Zebrafish entier Mont haute résolution double fluorescent In Situ Hybridation

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

Fluorescence In situ</emL&#39;hybridation> (FISH) du Protocole dans le sperme humain

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

Hybridation in situ pour la localisation précise des transcriptions dans les plantes

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

Radioactif In situ D&#39;hybridation pour détecter divers profils d&#39;expression génique dans les tissus

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

La détection de l&#39;ARN viral par fluorescence<em&gt; In situ</emHybridation&gt; (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

Timing chromosome Réplication Combiné avec fluorescent<em&gt; In situ</em&gt; Hybridation

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Toute Mont ARN fluorescent<em&gt; In situ</emHybridation&gt; d&#39;<em&gt; Drosophila</em&gt; Embryons

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

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 ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

Protéomique Préparation de l&#39;échantillon de formol et inclus en paraffine fixe tissus

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...