Ce projet a été financé par les IRSC et le CRSNG. Nous tenons à remercier Jusiak Barb pour contribuer à la création de souches exprimant volée DsRed étiquetés SSR (ligne BJ), et la UBC de bio-imagerie facilité.

Partie 1: Préparation des tissus <ol><li> Notre objectif est une préparation de tissu de larves de mouches, où le système nerveux est intact, mais la surface intérieure de la paroi musculaire du corps est exposé à une hémolymphe artificielle, et peut être positionné à proximité d&#39;un microscope pour lamelle de bonnes qualités optiques. En d&#39;autres termes, une préparation à l&#39;envers des larves. Depuis des préparations de tissus conventionnels au découpage, épinglant et étirement du muscle de la paroi du corps, et parfois enlever une partie du système nerveux, nous avions besoin d&#39;une approche différente. Nous voulions que les animaux à l&#39;intérieur-out parce que nous voulions obtenir un bon aperçu des caractéristiques structurelles et des changements structurels au cours du temps dans la synapse, et nous voulions garder le système nerveux intact. Nous voulions aussi éviter d&#39;étirer le tissu, et l&#39;activation de récepteurs d&#39;étirement, ce qui rendrait les muscles à contraction et la ruine de nos images. </li> <li> Pour commencer, le stade de l&#39;animal. Nourrir les larves troisième et errant larves tiers sont gros et faciles à décortiquer donc nous allons démontrer sur W3 larves. Cet animal est le phénotype a Tubby, et est large, de sorte qu&#39;il est facile d&#39;Evert. Nous avons seulement utilisé les larves que nous avons vu ramper activement, y compris les larves W3. </li><li> Nettoyer la surface d&#39;une mouche avec un pinceau très doux dans une boîte de Petri de H2O bidistillée. Nettoyer les larves ont une meilleure optique et de nettoyage réduit les bactéries. </li><li> Transfert de l&#39;animal à une petite boîte de Petri d&#39;environ 3 ml de glace froide HL-6, et l&#39;hémolymphe artificielle. Mettez le plat sur la glace jusqu&#39;à ce que l&#39;animal cesse de bouger, et détend (environ 5 minutes). </li><li> Tenez pince pointe très fine dans une main et les ciseaux à ressort dans l&#39;autre. J&#39;utilise les ciseaux avec ma main dominante. Faire un petit trou dans la paroi du corps avec les ciseaux pour equlibrate la pression à travers la paroi du corps. </li><li> Tenir l&#39;animal en douceur avec la pince vers le bas sur le fond du plat et couper les deux segments postérieurs. Disséquer loin les viscères et la graisse corporelle sera probablement de sortir de la cavité du corps. Coupez ce tissu aussi. </li><li> Tenir les larves contre le fond plat avec une pince fine. Tenir une broche # 0 insecte (avec une pointe émoussée) et poussez-le contre les pièces buccales de la larve. Poussez les pièces buccales dans la cavité du corps, comme vous tournez une chaussette à l&#39;envers. </li><li> L&#39;intérieur des tissus va ressembler à la figure: ci-dessous. Avec l&#39;outil ultra-fine dissection, disséquer le cadavre de graisse et de la trachioles paroi du corps. Essayez vraiment difficile de ne pas tirer la trachioles, ou déconnecter le système nerveux. Ripping l&#39;trachioles va déchirer les trous dans la paroi musculaire du corps. Retirer le corps autant de gras que vous pouvez. Ces deux structures perturber la qualité optique de votre tissu. Vous pouvez sauter le café avant de faire cette préparation. </li> <li> Lorsque vous avez terminé le muscle sera translucide, pas opaque ou blanc. Si vous mettez la préparation de tissu dans HL-6 sans glutamate, à température ambiante, il sera probablement parce que le contrat de façon rythmique générateurs schéma moteur dans le SNC sont travail. Évitez d&#39;utiliser des préparations avec des muscles du corps mur évidemment contractés, ou irrégulièrement contractés. L&#39;intérieur des murs intacts corps tend facilement à plier en deux le long de la lignes médianes dorsale et ventrale, alors la prépa donne un droit ou gauche &quot;hémi-animal» à visualiser. </li><li> 1.9 Monter les tissus soit dans une chambre de petit volume commercial, comme la chambre de Warner, ou une lame de microscope avec un arrangement lamelle comblé. Voir la partie 3 pour obtenir des suggestions sur le tissu de montage. </li></ol> Partie 2: Étiquetage Bouton neuronale avec l&#39;anticorps primaire marqué par fluorescence contre la HRP. <ol><li> Mettez 50 litres micro de marquage fluorescent anti-corps contre les primaires de HRP dans HL-6 dans une goutte sur une boîte de Pétri. Plonger la préparation dedans-dehors du corps mur dans le bain de teinture. Vous pouvez voir l&#39;étiquetage terminale neurone après environ 5 minutes, mais incuber pendant 10-20 minutes, pour une étiquette complète et claire. </li><li> Rincer le colorant pendant 10-30 secondes dans HL-6 à la température ambiante. Non liés colorant se rince rapidement, de sorte que le cycle de rinçage ne doivent pas être agressifs. </li><li> Varier la concentration de colorant et un temps d&#39;incubation nécessaire. </li></ol> Partie 3: étiquetage espace Perisynaptic de dextran conjugué flurophore <ol><li> Diluer fluorescence conjugué dextrane colorant dilué dans HL-6. Une concentration qui a bien fonctionné pour nos besoins a été: </li><li> Si vous n&#39;êtes pas préoccupés par le calendrier de l&#39;accès à votre colorant dans l&#39;espace extracellulaire, mettre une goutte 20 micro litres de colorant sur un &quot;pont&quot; lamelle (voir partie 4) et le dépôt de la préparation dans le bain de teinture. </li></ol> Partie 4: Montage du tissus pour la visualisation (avec la microscopie confocale). Si vous n&#39;avez pas besoin de perfuser votre préparation (observations court) ou que vous voulez garder le volume de la baignade HL-6 petits, utilisez la méthode glisser à double pont Si vous voulez perfuser votre préparation, essayez d&#39;utiliser une chambre de perfusion. Nous avons utilisé un moChambre à partir dified Instruments Warner. Détails à la fois pour suivre. Montage de préparation sur une lame à double pont. <ol><li> Utilisez une lame de microscope très propre. Superglue deux carrés, 18mm (# 1.5) des lamelles sur la lame. Laisser un écart de 2 mm entre les bords de la lamelle. </li><li> Laissez la colle sécher complètement, ou il forme un film bizarre sur le milieu aqueux autour de votre préparation. </li><li> Position à l&#39;intérieur des larves entre les bords de la lamelle. Vous pouvez avoir besoin de positionner la prépa sur une diagonale si les larves sont de grandes et de votre système d&#39;acquisition d&#39;image a une matrice de pixels limitée. </li><li> Couvrir le tissu avec une lamelle # 1.5, 18mm (très propre). Respecter le &quot;top&quot; lamelle la diapositive apposé glisse tissus d&#39;accompagnement, avec de la vaseline minime. </li><li> Mettre une goutte d&#39;huile Cargill objectif personnalisé avec un indice de réfraction de 1,3379 (si vous utilisez HL-6) sur la lamelle supérieure et flip cette assemblée plus. </li><li> Positionner l&#39;ensemble des tissus de votre côté du pétrole microscope, vers l&#39;objectif, et de concentrer votre objectif. Montage du tissu dans une chambre de perfusion modifié. </li><li> Collez une lamelle de 1,5 à former un plancher dans la chambre de RC 20. Placez le tissu dans la chambre que par RC-20 des instructions. Utilisez un morceau de filet Nytex au lieu d&#39;une lamelle pour former un toit pour la chambre. </li></ol> Partie 5: Résultats du représentant: <ol><li> Inside-out séquence dissection des tissus de préparation: <img src="/files/ftp_upload/1154/fig1.jpg" alt="Figure 1" /> W3 larves, lavé (&quot;Tubby&quot; phénotype) (5.1.1). W3 larves, postérieure 2 segments disséqués. Vous pouvez voir le corps de graisse poussé hors de la cavité du corps par voie trans-corps de pression mur (flèche). Essayez de minimiser viscérale &quot;éruption&quot; en faisant un petit trou dans le mur de corps 1 minute avant la dissection (5.1.2). La préparation avec les viscères disséqué avant de tourner la préparation à l&#39;envers (5.1.3). La préparation, la plupart du temps éversée, avec l&#39;axe (flèche) à l&#39;intérieur du lumen de la prépa (5.1.4). Le muscle est maintenant à l&#39;extérieur et la cuticule est à l&#39;intérieur, avec un corps gras et trachioles attachés (5.1.5). Une préparation des tissus entièrement disséqué avec presque tous les corps gras et trachioles disséqué (5.1.6). <img src="/files/ftp_upload/1154/fig2a.jpg" alt="Figure 2" width="600" height="200" /></li><li> Direct étiquetage utilisant des anticorps anti-HRP à la JNM larvaire. Un représentant W3 larvaires nerf-muscle synapse avec une extension gliales (intérieur vers l&#39;extérieur-prep). Le neurone moteur terminaux bouton sont marqués avec un primaire anti-corps contre HRP (magenta), qui est conjugué à Cy5. Le processus gliales sont étiquetés avec la GFP (green). <img src="/files/ftp_upload/1154/fig2b.jpg" alt="Figure 3" /></li><li> L&#39;espace Perisynaptic fluorescence visualisées avec Alexa 680 dextrane dans un intérieur vers l&#39;extérieur de préparation. Le processus gliales (vert) est étiqueté avec la GFP membrane cible. La SSR post-synaptique sur la surface du muscle (en bleu) est étiqueté avec DsRed. L&#39;Alexa dextrane (rouge) se concentre dans les zones les formes extracellulaires. Le colorant dextrane formes des piscines en forme de beigne dans les espaces perisynaptic. L&#39;étiquetage de dextran et DsRed SSR étiquetés sont présentés en niveaux de gris. Notez les piscines multiples beignets en forme de teinture mettant en évidence les espaces perisynaptic (flèche). </li></ol>

<ol>
	<li>Macleod, G. T., Marin, L., Charlton, M., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+Vesicles:+Test+for+a+role+in+presynaptic+Calcium+regulation.">Synaptic Vesicles: Test for a role in presynaptic Calcium regulation.</a> J. Neurosci. 24, 2496-2505 (2004).</li><li>Macleod, G. T., Hegstro, M., Wojtowicz, M., Charlton, M. P., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+Calcium+signals+in+Drosophila+motor+neuron+terminals.">Fast Calcium signals in Drosophila motor neuron terminals.</a> J. Neurophysiology. 88, 2659-2663 (2002).</li><li>Morales, M., Ferrus, A., Martinez-Padron, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+calcium-channel+currents+in+normal+and+csp+mutant+Drosophila+peptidergic+terminals.">Presynaptic calcium-channel currents in normal and csp mutant Drosophila peptidergic terminals.</a> Eur J Neurosci. 11, 1818-1826 (1999).</li><li>Stork, T., Engelen, D., Krudewig, A., Silies, M., Bainton, R. J., Kla&#168;mbt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+Function+of+the+Blood&#8211;Brain+Barrier+in+Drosophila.">Organization and Function of the Blood&#8211;Brain Barrier in Drosophila.</a> J. Neurosci. 28, 587-597 (2008).</li></ol>

Cette procédure permet à long terme d&#39;imagerie de vivre protéines marquées et les processus cellulaires. La préparation de tissu in situ, nous avons décrit est une SNC intacte et son fonctionnement, PNS et circuits réflexes. Cette préparation de tissus a des avantages sur la norme des larves protocoles musculaire volée, où le muscle du corps larvaire mur est tendu (quand il est tombé sur). Les étirements peuvent fausser la morphologie synaptique et de déclencher des contractions réfle...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>HL-6: Artifical Drosophila hemolymph, with 5 mM L-glutamate added, and 2 mM Calcium.</td>
<td>Reagent</td>
<td>NA</td>
<td>NA</td>
<td>5 mM L-glutamate blocked muscle contractions. We used Molecular grade L-Glutamate (Sigma).
2 mM Calcium is close to physiological Calcium levels in natural larval hemolymph.
References: Macleod et al 2002 and Macleod 2004
</td>
</tr>
<tr>
<td>Dextran, Alex Fluor 680; 10,000 MW, anionic, fixable</td>
<td>Reagent</td>
<td>Molecular Probes/Invitrogen</td>
<td>D34680</td>
<td>Use a small volume perfusion chamber to keep the total volume of dye low</td>
</tr>
<tr>
<td>Anti-HRP-CY5 conjugate (goat)</td>
<td>Reagent</td>
<td>Jackson ImmunoResearc</td>
<td>123-175-021</td>
<td>Dilute 2.0 mg into 1 ml ddH2O; aliquot into 4 microliter aliquots. Freeze at &#x2013;20C. Dilute one aliquot into 100 microliters of HL-6</td>
</tr>
<tr>
<td>Alexa 647 antibody labeling kit</td>
<td>Reagent</td>
<td>Molecular Probes/Invitrogen</td>
<td>A10475</td>
<td>We prepared a total of 80 micro liters of conjugated primary antibody, and stored as 2 microliter aliquots. We diluted each aliquot into 100 microliter of HL-6 for labeling.</td>
</tr>
<tr>
<td>Custom Formulated Objective Oil, refractive index 1.3379</td>
<td>Reagent</td>
<td>Cargill Labs</td>
<td>Custom Formulated</td>
<td>&nbsp;</td>
<tr>
<td>Ultra Fine Forceps</td>
<td>Tool</td>
<td>Fine Science Tolls</td>
<td>11252-23 or 11295-20</td>
<td>&nbsp;</td>
<tr>
<td>Spring scissors</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>91500-09</td>
<td>&nbsp;</td>
<tr>
<td>Ultra fine clipper scissors</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>15200-00</td>
<td>&nbsp;</td>
<tr>
<td>Perfusion Chamber RC 20 Series</td>
<td>Tool</td>
<td>Warner Instruments</td>
<td>64-02222</td>
<td>&nbsp;</td>
<tr>
<td>Spinning Disc confocal</td>
<td>Microscope</td>
<td>Quorum</td>
<td>Quorum Wave FX</td>
<td>Mounted on a Leica DMI6000 Inverted Microscope</td>
</tr>		</tbody>
		</table>
		</div>

visualizing the live drosophila glial-neuromuscular junction with fluorescent dyes

Notre projet a identifié GFP étiqueté structures gliales à la volée des larves synapse neuromusculaire développement. Pour regarder le développement de vivre gliales-nerf-muscle synapses, nous avons développé une préparation des tissus larvaires qui avait les caractéristiques des larves intactes vivre, mais a également eu de bonnes propriétés optiques. Cette nouvelle préparation a également permis l&#39;accès des perfusates vers la synapse. Nous avons utilisé des larves de mouches, les immergé dans l&#39;hémolymphe artificielle, et assoupli leurs contractions normales du corps rythmés par les paralysant. Ensuite, nous disséqué les segments postérieurs de chaque animal et des insectes avec une épingle émoussée poussé les parties de la bouche vers l&#39;arrière dans la cavité du corps. Cette éversée la paroi du corps larvaire, comme tourner une chaussette à l&#39;envers. Nous avons terminé la dissection aux ciseaux de dissection ultra-fines et donc exposé le côté viscéral du muscles de la paroi du corps. Les structures gliales à la JNM exprimé la GFP membrane cible sous le contrôle de promoteurs spécifiques gliales. La membrane post-synaptique, la SSR (Reticula Subsynaptic) dans le muscle ciblé exprimé synaptiquement DsRed. Nous avions besoin d&#39;acuité l&#39;étiquette des bornes du neurone moteur, la troisième partie de la synapse. Pour ce faire, nous avons appliqué aux anticorps primaires HRP, conjugué à un flurophore émettant rouge lointain. Pour tester les propriétés de diffusion du colorant dans l&#39;espace perisynaptic entre les bornes du neurone moteur et la SSR, nous avons appliqué une solution de molécules de dextran conjugué à grande flurophore émettant rouge lointain et les images collectées.

Watch this Scientific Journal Video about Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes at JoVE.com

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Nous avons décrit les caractéristiques structurelles des synapses neuromusculaires Glia-dans un roman de préparation des tissus intérieur-extérieur de larves de mouches en direct en utilisant des colorants fluorescents en microscopie confocale. Nous avons étiqueté terminaisons neuronales vivre avec fluorescentes anticorps primaires à HRP, et également visualisé l&#39;espace perisynaptic avec dextranes fluorescents.

Visualiser le Live Drosophile Gliales-jonction neuromusculaire avec des colorants fluorescents

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

visualizing-live-drosophila-glial-neuromuscular-junction-with

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Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Skeletal Muscle Relaxants

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10.7K vues.  University of British Columbia - UBC. Nous avons décrit les caractéristiques structurelles des synapses neuromusculaires Glia-dans un roman de préparation des tissus intérieur-extérieur de larves de mouches en direct en utilisant des colorants fluorescents en microscopie confocale. Nous avons étiqueté terminaisons neuronales vivre avec fluorescentes anticorps primaires à HRP, et également visualisé l'espace perisynaptic avec dextranes fluorescents.

Vidéo: Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Étiquetage des cellules souches avec des colorants fluorescents pour les non-invasive de détection avec l&#39;imagerie optique

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

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The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

La préparation des Drosophile Les embryons destinés à Live-imagerie utilisant le protocole goutte suspendue

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

Marquage fluorescent Drosophile</emOuvrages d&#39;art> Coeur

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

Visualiser le cœur battant Drosophile</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

Visualisation des bactéries dans nématodes en utilisant la microscopie à fluorescence

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Vivre imagerie de dédouanement cellulaire par apoptose au cours<em&gt; Drosophila</em&gt; Embryogenèse

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Visualisation Fréquence Stromule avec Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

Mesure simultanée de mitochondrial Calcium et mitochondrial potentiel membranaire dans des cellules vivantes par microscopie à fluorescence

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

Analyse du Gap Junction-dependent Transfert de miARN avec microscopie 3D-FRAP

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

Visualisation et suivi des ARNm endogènes dans Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...