Les auteurs tiennent à remercier le Dr Louis Reichardt pour les souris TrkA taulacZ et Dr. Annette Markus pour la discussion et suggestions perspicaces. Ce travail a été soutenu par des fonds de démarrage de la Fondation Burke ainsi que la Fondation Whitehall subvention de recherche 2010-08-61, une subvention de la Fondation Wings de vie (WFL-US-028/14) pour la recherche, accorder ZB1-1102-1 de la Christopher et Dana Reeve Foundation, et les subventions et 1R01EY022409 3R01EY022409-01S1 du National Eye Institute, à JZ. KJO est un compagnon de Goldsmith.

NOTE: Toutes les procédures sont conformes à la NIH Guide pour l&#39;entretien et utilisation des animaux de laboratoire. Le protocole d&#39;animal a été approuvé par le IACUC au Weill Cornell Medical College. 1. Préparation des tissus <ol><li> Euthanasier femelles chronométré-grossesse par dislocation cervicale 15. Disséquer embryonnaires E16 - E18 embryons de femelles chronométré-grossesse et placer des embryons individuellement dans les puits d&#39;une plaque à 6 puits, remplis de solution saline tamponnée phosphate froide (PBS). </li><li> Rincer embryons dans du PBS froid. </li><li> Retirer une petite partie de la membrane amniotique de l&#39;embryon et le placer dans une solution pré-préparés protéinase K pour le génotypage ultérieure. Alternativement, si la membrane amniotique est perdu, enlever une petite partie de la pointe de la queue de l&#39;embryon et le placer dans une solution de proteinase K </li><li> Percez des trous dans la peau tout autour de l&#39;embryon en utilisant des épingles insectes (au moins 100 par côté). </li><li> Retirer du PBS et ajouter lentement frais 2% de paraformaldéhyde (PFA),pH 7,4, au puits. </li><li> Agitez doucement pendant 2 h à 4 ° C. </li><li> Rincer les embryons à deux reprises dans du PBS et les stocker dans du PBS à 4 ° C jusqu&#39;à ce que la coloration. </li></ol> 2. génotypage <ol><li> Plongez membranes amniotiques ou queues d&#39;embryons dans la protéinase K mélange selon les instructions du fabricant. </li><li> Incuber à 56 ° C pendant 10 min. </li><li> Extraire l&#39;ADN en utilisant un kit de purification d&#39;ADN disponible dans le commerce. </li><li> Prendre 0,5 ul du total 200 ul purifiés solution d&#39;ADN génomique, le combiner avec 0,5 pl de chacune des amorces sens et antisens spécifiques d&#39;un gène (10 uM), 2 ul de mélange dNTP (2,5 mM de dATP, dCTP, dGTP et dTTP) , 0,2 ul de polymerase de Taq (5 U / ul) et du tampon de PCR 2 pi (10x), ajoutez H 2 O à un volume total de 20 ul. NOTE: Les séquences des amorces sont les suivantes (tableau 1): TrkA: TrkA_F: GCG GGC GCC GCC GCG ATG, TrkA_R: GAA GCC GCC TGC GCG GCT CTG CCA GGG TG; Frag PCRlongueur ment: 400 paires de bases (pb). Bax: In5R: GTT GAC CAG AGT GGC GTA GG, Ex5F: TGA TCA GAA CCA TCA TG, NéoR: GCC CTT CCA TTG CTC AGC GG; PCR fragments longueurs: type sauvage, 304 pb; Bax null, 507 pb. TauLacZ: lacZup: ACA ACG TCG TGA GTG GGA AAA, lacZdown: ATC AAC ATT AAA TGT GAG CGA G; PCR de longueur des fragments: 360 pb. </li><li> Exécuter une PCR avec le programme suivant pour TrkA: étape 1: 1 min à 95 ° C, étape 2: 10 sec à 95 ° C, étape 3: 20 sec à 68 ° C, étape 4: 30 sec à 72 ° C. Répétez les étapes 2 à 4 pour 40 cycles. <ol><li> Pour LacZ et Bax, exécutez une PCR avec le programme suivant: étape 1: 1 min à 95 ° C, étape 2: 10 sec à 95 ° C, l&#39;étape 3: 1 min à 54 ° C, étape 4: 1 min à 72 ° C, répétez les étapes 2 à 4 pour 35 cycles. </li></ol></li><li> Exécuter les échantillons de PCR sur un gel d&#39;agarose à 2% à 100 V dans un tampon Tris-acétate 1x-EDTA pendant 20 min avec une voie de échelle d&#39;ADN pour évaluer la longueur des fragments. </li><li> Analyser embryons pour la présence de type sauvage TrkA, TRKA-tauLacZ et Bax allèles nuls. Les génotypes des embryons expérimentaux sont TrkA taulacZ / WT: Bax - / - TrkA et taulacZ / taulacZ: Bax - / -, en fonction des questions expérimentales. Les embryons qui ne ont pas le journaliste tauLacZ seront négatifs pour axonale coloration et peuvent donc servir de témoins négatifs pour calibrer le processus de coloration. </li></ol> 3. embryon coloration <ol><li> Utilisez un kit commercial pour lacZ coloration avec quelques modifications telles que décrites ci-dessous dans les sections 3.2 à 3.8. Ici, utiliser le kit Millipore. </li><li> Plongez embryons dans le tampon A pendant au moins 1 h, secouant doucement à la température ambiante. </li><li> Immerger directement embryons dans le tampon B pendant au moins 15 minutes, en secouant doucement à la température ambiante. </li><li> Lorsque des embryons sont dans le tampon A / B, solution de pré-base chaude de tissus à 37 ° C. Utilisation 5 ml de solution de base de tissu par embryon. </li><li> Lorsque des embryons sont dans le tampon A / B, préparer une nouvelle 5-bromo- 4- chloro- 3- indolyle α-D-galactopyranoside (X-gal) à une concentration de 40 mg / ml dans 100% de diméthylsulfoxyde (DMSO). </li><li> Diluer X-gal dans la solution préchauffée base de tissu à une concentration de 1 mg / ml et ajouter 5 ml de X-gal / le mélange de base de tissu à un flacon à scintillation en verre. </li><li> Transfert d&#39;embryons dans un flacon à scintillation en verre contenant le mélange de base X-gal / tissus. Sceller le couvercle avec du Parafilm et incuber à 37 ° C pendant la nuit, la vérification de présence de bleuissement. </li><li> Embryons de Postfix avec frais PFA 4%, pH 7,4. Agitez doucement pendant 4 heures à 4 ° C. </li><li> Rincer embryons deux fois dans PBS froid. </li></ol> 4. tissus déshydratation et de compensation <ol><li> La place embryons dans 50% de méthanol / 50% d&#39;un mélange PBS pendant 30 min à température ambiante en agitant dans les flacons de verre. </li><li> Continuer à déshydrater dans 75% de méthanol / 25% de PBS pendant 30 min à température ambiante en agitant dans des flacons en verre. </li><li> Continuer à se déshydrater dans 90% de méthanol / 10% PBS pendant 30 min à température ambiante en agitant dans des flacons de verre. </li><li> Continuer à déshydrater dans 100% de methanol pendant 30 min (2x) à la température ambiante en agitant dans des flacons en verre. </li><li> Lorsque des embryons sont déshydratant, préparer un mélange 1: 1 (v: v) un mélange d&#39;alcool de benzoyle et benzoyle benzoate. REMARQUE: l&#39;alcool de benzoyle et benzoyle benzoate est toxique. </li><li> Immerger les embryons dans le complexe 1: 1 (v: v) mélange de methanol à 100%: 100% BABB pendant 15 min. Utilisez uniquement parce que le verre BABB va se dissoudre en plastique. </li><li> Plongez embryons dans 100% BABB effacer complètement le tissu et de visualiser coloration X-gal dans l&#39;embryon effacé. REMARQUE: Image dès que possible parce que la tache X-gal va se estomper au fil du temps. </li></ol> 5. Mont entiers Imaging <ol><li> Stabiliser l&#39;embryon, immergé dans 100% BABB, aux angles appropriés pour la photographie utilisant des pinces métalliques. Illuminez utilisant une combinaison de sources jusqu&#39;à la lumière et sous-lumière lumière. </li><li> Acquérir des images avec un microscope disséquantéquipé d&#39;un appareil photo numérique. Prenez plusieurs expositions aux champs lumineux à cinq plans focaux successives 0,5 mm d&#39;intervalle le long de l&#39;axe Z. Les temps d&#39;exposition et diaphragmes peuvent varier en fonction de l&#39;éclairage et de la caméra spécifications et doivent être optimisés pour chaque configuration. </li><li> Utilisez un logiciel de traitement d&#39;image pour traiter des images de superposition et de générer des photomontages. </li></ol>

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The authors declare that there are no competing interests.

Le X-gal procédure de souris embryonnaires TrkA taulacZ coloration décrite ci-dessus permet la visualisation rapide et détaillée des projections axonales longue distance dans l&#39;embryon fixe intacte. En raison de la Bax fond nul ces souris permettent de sonder des mécanismes qui peuvent contribuer à la croissance de l&#39;axone et la survie neuronale de signalisation. Accouplement avec transgéniques ou knock-out souris d&#39;intérêt permet une évaluation globale des phénotypes ...

Mise en place des réseaux neuronaux précis est un processus complexe du développement essentiel pour le fonctionnement du système nerveux. La perturbation de ce processus conduit à un dysfonctionnement neuronal qui a été impliquée dans des maladies neurologiques humaines 3.1. Pour étudier les mécanismes moléculaires sous-jacents de la croissance axonale et la cible innervation chez les mammifères, nous avons développé un protocole de visualiser les trajectoires axonales de TrkA exprimant neurones sensoriels utilisant une combinaison de deux lignées de souris génétiquement modifiées. TrkA est un récepteur pour le facteur NGF de croissance des nerfs et est un marqueur fonctionnel des neurones sensoriels nociceptifs 4. TrkA est fortement exprimé dans les neurones nociceptifs au cours du développement précoce et la médiation de la survie des neurones dépendant du NGF, la croissance des axones, arborisation cible innervation et 9.5. Chez les souris TrkA taulacZ, le gène de type sauvage TrkA est remplacé par une expression de c taulacZassette 10, de telle sorte que la morphologie axonale des neurones positifs putatifs TrkA peut être visualisée par β-gal (X-gal) 11 coloration. Utilisation d&#39;une ligne TrkA taulacZ / WT hétérozygote, nous pouvons examiner les facteurs qui peuvent réglementer ou interférer avec le développement de projections sensorielles afférentes in vivo. En outre, l&#39;expression TrkA est absent chez les souris homozygotes TrkA taulacZ / taulacZ, qui peuvent donc être utilisés pour évaluer la croissance de l&#39;axone promouvoir des mécanismes en l&#39;absence de signalisation du NGF / TrkA. Etant donné que les neurones nociceptifs dépendent de NGF / TrkA signalisation non seulement pour la croissance des axones, mais aussi pour la survie, on emploie une autre lignée de souris, dépourvu du gène Bax pro-apoptotique, pour inhiber l&#39;apoptose dans les neurones de DRG embryonnaires, les sauver de la mort cellulaire qui est par ailleurs observé en l&#39;absence de signalisation TrkA. Le Bax - / - fond 12 permet ainsi pour la molécudissection lar des voies qui affectent spécifiquement 7-9,13-15 de croissance axone signalisation. Dans TrkA - / -: - Bax / - souris, les neurones DRG survivre, mais sensorielle innervation afférente dans la peau est complètement abolies 14,15. Nous pouvons activer sélectivement les voies de signalisation pour déterminer leurs contributions respectives à l&#39;élaboration de projections axonales. L&#39;utilité de cette méthode est qu&#39;elle permet à l&#39;évaluation des changements de phénotypes de croissance axonale lorsque différentes modifications génétiques sont élevés sur le TrkA taulacZ / taulacZ: Bax - / - ou TrkA taulacZ / WT: Bax - / - milieux.

<table border="0" cellpadding="0" cellspacing="0" width="465">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;"></td>
			<td style="width:127px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PFA</td>
			<td>Sigma-Aldrich</td>
			<td>P6418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Life Tech</td>
			<td>10010-023</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Rinse Solution A</td>
			<td>Millipore</td>
			<td>BG-6-B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Rinse Solution B</td>
			<td>Millipore</td>
			<td>BG-7-B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Stain Base Solution</td>
			<td>Millipore</td>
			<td>BG-8-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-gal&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>B4252</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass scintiallation vial</td>
			<td>Kimble Chase</td>
			<td>74500-20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td>Labline</td>
			<td>Model 120</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insect pins</td>
			<td>FST</td>
			<td>26000-30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 well dish</td>
			<td>USA Scientific</td>
			<td>CC7672-7506</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primers</td>
			<td>IDT</td>
			<td>custom DNA primers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Takara dNTP mixture</td>
			<td>Takara</td>
			<td>4030</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Takara LA buffer</td>
			<td>Takara</td>
			<td>RR002A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Takara LA Taq</td>
			<td>Takara</td>
			<td>RR002A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR machine</td>
			<td>Bio-Rad&#160;</td>
			<td>DNA Engine Dyad</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>B-1042</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl benzoate</td>
			<td>Sigma-Aldrich</td>
			<td>B-6630</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope</td>
			<td>Leica</td>
			<td>M205A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Camera</td>
			<td>Leica</td>
			<td>DFC310FX</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ring light&#160;</td>
			<td>Leica&#160;</td>
			<td>MEB110</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Photoshop</td>
			<td>Adobe</td>
			<td>Photoshop 4.0</td>
		</tr>
	</tbody>
</table>

whole-mount imaging of mouse embryo sensory axon projections

La visualisation de pleine longueur projections neuronales dans les embryons est essentiel d&#39;acquérir une compréhension de la façon dont les mammifères réseaux neuronaux se développent. Nous décrivons ici un procédé pour marquer in situ un sous-ensemble de ganglion de la racine dorsale (DRG) de projections axonales à évaluer leurs caractéristiques phénotypiques utilisant plusieurs lignées de souris génétiquement modifiées. Les neurones TrkA-positif sont des neurones nocicepteurs, dédiées à la transmission des signaux de douleur. Nous utilisons une lignée de souris TrkA taulacZ d&#39;étiqueter les trajectoires de tous les axones périphériques TrkA-positif dans l&#39;embryon de souris intactes. Nous élevons en outre la ligne TrkA taulacZ sur un fond nul Bax, qui abolit essentiellement apoptose neuronale, afin d&#39;évaluer les questions indépendamment de possibles effets de manipulations génétiques sur la survie neuronale liés à la croissance. Par la suite, les souris génétiquement modifiées d&#39;intérêt sont élevés avec le TrkA TaullacZ / Bax ligne nulle et sont alors prêts pour l&#39;étude en utilisant les techniques décrites ici. Cette présentation contient des informations détaillées sur les plans d&#39;élevage de souris, le génotypage au moment de la dissection, la préparation des tissus, la coloration et la compensation pour permettre la visualisation de pleine longueur trajectoires axonales dans l&#39;ensemble du montage préparation.

Les génotypes de TrkA WT / taulacZ: Bax - / - TrkA et taulacZ / taulacZ: Bax - / - embryons peuvent être déterminées sans ambiguïté par génotypage PCR standard (figure 1). Coloration X-Gal affiche des informations détaillées tonnelles axonales périphériques sous-cutanée chez les embryons classiquement colorées (figures 2, 3a), et dans tout l&#39;embryon après compensation de tissu (figures 3b, 4). <p class="j...

Watch this Scientific Journal Video about Whole-mount Imaging of Mouse Embryo Sensory Axon Projections at JoVE.com

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

Whole-monter imagerie de l&#39;embryon de souris projections sensorielles Axon

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

whole-mount-imaging-of-mouse-embryo-sensory-axon-projections

Research

JoVE Journal

Neuroscience

10.0K vues.  Weill Medical College of Cornell University. We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

Vidéo: Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Multiple-Mouse Imaging neuroanatomiques par résonance magnétique

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Recherche

Neurosciences

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

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

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

Dissection d&#39;un oeil de la souris pour un mont entiers de l&#39;épithélium pigmentaire rétinien

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

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Reproductible écrasement du nerf sciatique de souris et l&#39;évaluation subséquente de la régénération par l&#39;analyse du Mont muscle entier

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Mont entiers immunofluorescence de la rétine de souris néonatal chargé d&#39;enquêter sur l&#39;angiogenèse<em&gt; In vivo</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Tout le mont immunomarquage des récepteurs olfactifs neurones dans le<em&gt; Drosophila</em&gt; Antenne

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale - as seen, for example, in the orderly arrangement of cell types within a single hair follicle - and on a macroscopic scale - as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.

Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.

Appartement Mont imagerie de la peau de souris et son application à l&#39;analyse des follicules pileux Modélisation et sensorielle Axon Morphologie

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Imagerie Ca<sup&gt; 2+</sup&gt; Dynamics dans les terminaux cône photorécepteur Axon de la rétine de souris

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

Mont entiers Dissection et immunofluorescence de la cochlée de souris adultes

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Tomographie par cohérence optique : Imagerie souris ganglionnaires rétiniennes cellules In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

Immunomarquage quadruple de l&#39;ampoule olfactive pour la visualisation des codes d&#39;identité moléculaire des axes sensoriels olfactifs

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...