Les auteurs tiennent à remercier James Oesterling pour cytométrie de flux de l&#39;aide. Cette recherche a été financée par une subvention de l&#39;Institut national américain du cancer (R01-CA178380, ZY) et une subvention US National Institutes postdoctorale de formation (5T32CA009035-37, LWY).

L&#39;utilisation des animaux dans ce protocole a été effectué conformément aux procédures approuvées par le Comité Cancer Center soin et l&#39;utilisation des animaux Fox Chase. 1 Préparation des instruments, des solutions et Lamelles <ol><li> Nettoyer les deux ensembles de # 5 pinces fines, un ensemble de n ° 7 pince fine courbes, une grande ciseaux chirurgicaux, un ciseau de microdissecting, un spatule et une cuillère perforée. Placer les instruments dans une poche et autoclave de stérilisation auto-obturant. Gardez instruments de poche jusqu&#39;au moment de son utilisation. </li><li> Préparer une solution à 3% d&#39;agarose à basse température de fusion. <ol><li> Ajouter 1,5 g d&#39;agarose dans 50 ml de solution tampon phosphate stérile de Dulbecco (DPBS), puis placer au micro-ondes et de la chaleur jusqu&#39;à ce que des bulles apparaissent. ballon de tourbillon et réchauffer jusqu&#39;à ce que tous agarose a dissous. Mélanger la solution avec une barre d&#39;agitation avant le chauffage si touffes apparaissent. </li><li> Conserver la solution dans un bain-marie à 37 ° jusqu&#39;à ce que nécessaire. </li><li> Pour boît à long termee, stocker la solution d&#39;agarose pour une utilisation ultérieure à la température ambiante ou à 4 ° C pendant plusieurs mois. Réchauffer au micro-ondes pour liquéfier. Pour le chauffage répété de la solution d&#39;agarose, peser le ballon avant de chauffer pour maintenir son poids initial avec de l&#39;eau distillée stérile chaud. </li></ol></li><li> Préparer les solutions suivantes pour la dissociation des cellules sur la base de la papaïne-2 tel que décrit précédemment: <ol><li> Une solution de papaïne à la papaïne (100 U / ml), la cystéine (0,2 mg / ml) et de la DNase (250 U / ml) dans du phénol rouge stérile contenant du DPBS. </li><li> Une solution ovomucoïde avec de l&#39;albumine de sérum bovin (BSA; 8 mg / ml), un inhibiteur de la trypsine du soja (8 mg / ml)) et de la DNase (250 U / ml) dans du phénol rouge stérile contenant du DPBS. Stériliser par filtration des solutions et ajuster le pH à retourner couleur d&#39;origine du rouge de phénol contenant DPBS. </li></ol></li><li> Préparer des milieux de culture cellulaire (NB / B27): compléter milieux de base avec 1 mM de pyruvate de sodium, 2 mM de L-glutamine, 1% de pénicilline-streptomycine (P / S) et un supplément B27. Filter stériliser et protéger de la lumière. Equilibrer support à 37 ° C et 5% de CO 2 avant l&#39;utilisation. </li><li> Préparer le tampon FACS: 5% de sérum bovin fœtal (FBS) dans du DPBS. Ajouter 2,5 ml de FBS à 47,5 ml DPBS. </li><li> Préparation de poly-D-lysine (PDL) des lamelles revêtues d&#39;. <ol><li> Autoclave nouvelles lamelles de verre. </li><li> Enduire les lamelles avec PDL (100 ug / ml dans de l&#39;eau distillée stérile) et incuber pendant 2 heures à 37 ° C ou à température ambiante jusqu&#39;au lendemain. </li><li> Laver lamelles de d&#39;eau distillée stérile. Laver une fois avec NB / B27 médias. Laissez sécher complètement lamelles en culture de tissus capot avant de l&#39;utiliser. </li></ol></li></ol> 2 Dissection et préparation des tranches de tissu <ol><li> Le jour de dissection, essuyez la zone de dissection éthanol à 70% et la couverture d&#39;un tampon absorbant. </li><li> Déposer les outils de dissection de la poche de stérilisation. Ajouter glacée, DPBS stériles / 1% P / S à une petite boîte de Petri et placer sur la glace. </li><li> Décapiter P4 Math1-GFSouriceaux P / Nestin-PCP avec de grands ciseaux chirurgicaux détiennent alors la tête vers le bas avec de fines pinces courbes. Enlever la peau et peler doucement le crâne avec # 5 pinces fines puis évider le cerveau du crâne à l&#39;aide d&#39;une spatule et transférer à la boîte de Pétri contenant du DPBS / 1% P / S. <ol><li> Utilisation # 5 pinces fines, séparer soigneusement le cervelet du reste du cerveau. Assurez-vous de disséquer un chiot à un moment aussi rapidement que possible. </li><li> Recueillir un minimum de 8 cervelet afin d&#39;obtenir suffisamment de PES pour les analyses moléculaires et fonctionnelles. </li></ol></li><li> Remplir un moule mesurant l&#39;incorporation de 2 x 2 x 2 cm avec une solution à 3% d&#39;agarose à basse température de fusion. Assurez-vous que la température de l&#39;agarose a pas plus de 37 ° C. </li><li> Retirer l&#39;excès de liquide en tamponnant doucement cervelet sur un tissu de laboratoire. Passer cervelet dans le moule dans une position verticale. Placer le moule dans la glace afin de lui permettre de se solidifier rapidement. Utilisez ~ 4 cervelet par bloc. </li><li> Obtenir licoupes de tissus ving avec un vibratome de lame vibrante. Coupez le bloc d&#39;agarose contenant cerebella-avec une lame de rasoir. Coller le bloc d&#39;agarose à la plaque avec vibratome cervelet orienté dans une position verticale. </li><li> Remplissez le bac de la vibratome avec DPBS glacée stérile / 1% P / S et mettre de la glace en dessous. <ol><li> Couper 600 um sections à travers toute la longueur du cervelet. Recueillir sections du plateau de glace à l&#39;aide d&#39;une cuillère perforée. sections de place dans une boîte de Pétri remplie de DPBS / 1% P / S sur la glace. </li></ol></li></ol> 3. microdissection et Cell Dissociation <ol><li> Placez la boîte de Petri contenant des tranches de tissu sous une dissection au microscope à fluorescence. <ol><li> Séparez délicatement l&#39;EGL du reste de la section du cervelet en utilisant une pince fine en disséquant entre le PCP couche + moléculaire et la GFP + EGL (voir ligne en pointillés sur la figure 1). Faites attention de ne pas inclure une partie de la couche moléculaire, qui se traduira parcontamination de Nestin exprimant Bergmann cellules gliales. </li><li> Terminez cette étape aussi rapidement que possible et de garder le tissu sur la glace pour éviter la mort cellulaire. </li><li> Retirer le agarose entourant le tissu avant de procéder à l&#39;étape suivante. </li></ol></li><li> Digérer le tissu EGL microdissection à l&#39;aide d&#39;un protocole à base de papaïne-2 tel que décrit précédemment pour obtenir une suspension cellulaire unique. <ol><li> Re-suspendre les cellules dans un tampon FACS pour la collecte de cellules CFP-positifs via FACS. </li></ol></li></ol> 4. Tri cellulaire et placage <ol><li> Trier les cellules de la PCP fluorescence en utilisant un flux à grande vitesse stérile cytomètre contenant un filtre approprié PCP et prélever des cellules dans un tampon FACS sur la glace. Obtenir environ 100.000 PES de chaque cervelet P4. </li><li> Centrifuger les cellules à 300 g pendant 5 min. Utiliser des cellules immédiatement pour l&#39;analyse moléculaire ou la culture pour de nouvelles expérimentations. </li><li> Pour des cellules en culture, remettre en suspension les cellules dans préchauffé NB / B27 mdias et la plaque sur des lamelles PDL revêtus tels que décrits précédemment 2. </li></ol>

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Les auteurs n&#39;ont rien à divulguer.

Procédé de microdissection décrite ici est le premier procédé capable d&#39;isoler une population pure de cellules vivantes pour l&#39;utilisation dans des analyses moléculaires et fonctionnels. Comme l&#39;a démontré par Li et al. 9, PES obtenus par ce procédé peuvent être mises en culture et incorporés dans diverses expériences et en raison de leur haut degré de pureté, peuvent également être utilisés pour l&#39;analyse de puces à ADN. Il e...

Le cervelet est constitué de plusieurs couches de cellules, chacune contenant des types cellulaires distincts. Au cours du développement, la prolifération des précurseurs EGL contient des granules de neurones (PNB), tandis que la couche moléculaire et la couche de Purkinje contiennent neurones Bergmann gliales et de Purkinje, respectivement. Profondément dans le cervelet se trouve la substance blanche, qui contient des cellules souches neurales (NSC) et les oligodendrocytes 1. Sonic hedgehog (Shh) joue un rôle essentiel dans la régulation du développement du cervelet et en particulier, il favorise la prolifération des PNB par liaison à son récepteur Patched (Ptc), un régulateur négatif de la signalisation de Shh 2-4. Activation aberrante de signalisation de Shh génère médulloblastome (MB), la tumeur la plus fréquente de cerveau maligne chez les enfants 5,6. Modèles de souris transgéniques Ptc de MB mutants sont des outils puissants pour l&#39;étude de la MB et développent des tumeurs ressemblant MB humaine 7,8. L&#39;utilisation de cessouris, on a découvert que le PNB sont la cellule d&#39;origine pour hérisson de type MB 7. En outre, en plus de PNB, nous avons récemment identifié une population unique de progéniteurs neuronaux au sein de l&#39;EGL du cervelet développement qui peut également donner lieu à MB. Ces cellules expriment des niveaux élevés de la protéine de type VI de filament intermédiaire, la nestine, et sont appelés PES 9. PES sont situés dans la partie profonde de la EGL et existent de façon transitoire seulement au cours du développement du cervelet néonatale. Ils se sont engagés à la lignée de cellules granulaires comme le PNB, mais sont distincts car ils sont au repos et n&#39;expriment pas Math1, un marqueur bien établi de PNB classiques 10. En outre, les PES donnent lieu à MB plus efficacement que le PNB après l&#39;activation de la voie de signalisation de Shh 9, qui en fait un roman pour origine MB tumorigenèse fait. Nous avons déjà isolé PES de l&#39;EGL cérébelleuse à jour postnatal 4 (P4) par la technique de microdissectiondécrit ici 9. Microdissection par visualisation microscopique direct du tissu permet de dissection spécifique de l&#39;EGL cérébelleuse. Cela est nécessaire car Nestin est également exprimé par les CNS dans la substance blanche du cervelet et par Bergmann cellules gliales dans la couche moléculaire 1,7,11,12 et il est essentiel que ces cellules ne sont pas inclus, car ils confondent l&#39;analyse. Des cellules isolées à partir de tissu microdissection peuvent être utilisés immédiatement pour l&#39;analyse moléculaire ou ils peuvent être cultivés pour d&#39;autres applications. Pour aider à microdissecting spécifiquement l&#39;EGL et pour purifier davantage les PES, les souris Math1-GFP (protéine fluorescente verte) ont été croisées avec des souris Nestin-CFP (cyan fluorescent protein). Le facteur de transcription Math1 est spécifiquement exprimée par le PNB et l&#39;expression de la GFP est clairement visible dans le EGL 9,13. Souris Nestin-PCP expriment une forme nucléaire de la PCP et peuvent être facilement visualisées dans la couche moléculaire 9,14. Ensemble, GFP et l&#39;expression de la PCP créent des frontières pour microdissection de la EGL (voir Figure 1). PES CFP-positifs ont ensuite été isolées par FACS, après la dissociation enzymatique des RAT disséqués. Actuellement, la méthode la plus utilisée à bon escient pour isoler les cellules de l&#39;EGL est gradient de Percoll centrifugation du tissu cérébelleux ensemble 15,16. Cette méthode, cependant, ne peut pas exclure totalement populations de cellules nestine + de la couche moléculaire et la substance blanche et ne peut donc pas être utilisé pour l&#39;étude de la PNI. Microdissection par capture laser, qui utilise le transfert d&#39;énergie laser pour retirer les cellules d&#39;intérêt, est un autre procédé utilisé pour isoler des cellules spécifiques au sein d&#39;un tissu hétérogène 17,18 cellules capturées mais ne peuvent être utilisés pour la récupération de l&#39;ADN, de l&#39;ARN et de la protéine et ne sont pas pouvoir être cultivées. Ce protocole fournit un moyen d&#39;isoler spécifiquement, une population pure vie des PES de l&#39;EGL.Cette technique peut également être appliquée à différents types de tissus qui ont soit l&#39;architecture reconnaissable anatomique ou marquées par fluorescence de cellules / régions. Par conséquent, le principal avantage de l&#39;intégration de cette nouvelle technique de microdissection dans les protocoles d&#39;isolement des cellules est que les cellules peuvent être isolées à partir de régions spécifiques du tissu pour éliminer les tissus voisins et de contaminer les cellules peuvent être recueillies immédiatement pour l&#39;analyse en culture ou pour d&#39;autres applications.

<table border="0" cellpadding="0" cellspacing="0" width="572">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Neurobasal media</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">21103-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">PDL</td>
			<td style="width:71px;">Millipore</td>
			<td style="width:119px;">A-003-E</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">ultra pure low melting temperature agarose</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">16520-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">DPBS</td>
			<td style="width:71px;">Gibco</td>
			<td align="right" style="width:119px;">14190144</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of distinct cell populations from the developing cerebellum by microdissection

Microdissection est une nouvelle technique qui peut isoler des régions spécifiques d&#39;un tissu et d&#39;éliminer la contamination à partir de sources cellulaires dans les zones adjacentes. Cette méthode a été utilisée la première fois dans l&#39;étude de progéniteurs exprimant la nestine (PES), une population de cellules nouvellement identifié dans la couche externe du cervelet germinal (EGL). Utilisation de microdissection en combinaison avec un tri cellulaire activé par fluorescence (FACS), une population pure de PES a été collecté séparément de précurseurs neuronaux granulés classiques de l&#39;EGL et d&#39;autres cellules exprimant la nestine-contaminantes dans le cervelet. Sans microdissection, des analyses fonctionnelles de PES n&#39;auraient pas été possible avec les méthodes actuellement disponibles, tels que gradient de Percoll centrifugation et microdissection laser. Cette technique peut également être appliquée pour une utilisation avec divers tissus qui contiennent des régions reconnaissables ou des cellules marquées par fluorescence. Plus important encore, un avantage majeur de cette microdissection technique est que les cellules isolées vivent et peuvent être cultivées pour plus d&#39;expérimentation, qui n&#39;est actuellement pas possible avec d&#39;autres méthodes décrites.

Comme le montre la figure 1A, tranches de cervelet ont été préparés à partir de / souris Nestin-PCP P4 Math1-GFP, dans lequel le EGL cérébelleux a été dominée par PNB exprimant la GFP et la couche moléculaire a été enrichi par les cellules gliales CFP-positifs. Pour isoler les PES qui sont situés dans la partie profonde de la EGL, tranches ont été microdisséquées entre l&#39;EGL et la couche moléculaire (le long de la ligne pointillée, figure 1A). R...

Watch this Scientific Journal Video about Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection at JoVE.com

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

progéniteurs exprimant la nestine sont une population nouvellement identifié des progéniteurs neuronaux dans le cervelet en développement. En utilisant la technique de microdissection présentés ici, en combinaison avec un tri cellulaire activé par fluorescence, cette population cellulaire peut être purifié sans contamination par d&#39;autres régions du cervelet et peut être mis en culture pour des études ultérieures.

Isolement des populations cellulaires distinctes du cervelet en développement par microdissection

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. ...

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9.5K vues.  Temple University Health System.  progéniteurs exprimant la nestine sont une population nouvellement identifié des progéniteurs neuronaux dans le cervelet en développement. En utilisant la technique de microdissection présentés ici, en combinaison avec un tri cellulaire activé par fluorescence, cette population cellulaire peut être purifié sans contamination par d'autres régions du cervelet et peut être mis en culture pour des études ultérieures. 

Vidéo: Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

Populations cellulaires Caractérisation électrophysiologique des exprimant la GFP dans la rétine intacte

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

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Neurosciences

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Isolement et culture de cellules de crêtes neurales de tube neural embryonnaire murin

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

Microdissection par capture laser de populations enrichies de neurones neurones isolés ou pour l&#39;analyse d&#39;expression génique après un traumatisme crânien

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

Production d’ARN pour l’analyse transcriptomique à partir de corps cellulaires de motoneurones de la moelle épinière de souris par microdissection de capture laser

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Imagerie en direct de la mitose dans les développement Cortex souris embryonnaires

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

Microdissection laser - une démonstration de l&#39;isolement de la dopamine neurones individuels et de l&#39;Espace ventrale tegmentale entière

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

Vivre l’imagerie suivie d’une cellule unique de suivi pour surveiller la biologie cellulaire et la Progression de la lignée de plusieurs Populations de neurones

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

Optimisation de Microdissection Laser-Capture d’isolement de Ganglia entérique de tissu humain frais-congelé

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or regions from heterogenous tissues. Captured product can be used in a variety of molecular methods for protein, DNA or RNA isolation. However, preservation of RNA from postmortem human brain tissue is especially challenging. Standard visualization techniques for LCM require histologic or immunohistochemical staining procedures that can further degrade RNA. Therefore, we designed a stainless protocol for visualization in LCM with the intended purpose of preserving RNA integrity in post-mortem human brain tissue. The Purkinje cell of the cerebellum is a good candidate for stainless visualization, due to its size and characteristic location. The cerebellar cortex has distinct layers that differ in cell density, making them a good archetype to identify under high magnification microscopy. Purkinje cells are large neurons situated between the granule cell layer, which is a densely cellular network of small neurons, and the molecular layer, which is sparse in cell bodies. Because of this architecture, the use of stainless visualization is feasible. Other organ or cell systems that mimic this phenotype would also be suitable. The stainless protocol is designed to fix fresh-frozen tissue with ethanol and remove lipids with xylene for improved morphological visualization under high magnification light microscopy. This protocol does not account for other fixation methods and is specifically designed for fresh-frozen tissue samples captured using an ultraviolet (UV)-LCM system. Here, we present a full protocol for sectioning and fixing fresh frozen post-mortem human cerebellar tissue and purification of RNA from Purkinje cells isolated by UV-LCM, while preserving RNA quality for subsequent RNA-sequencing. In our hands, this protocol produces exceptional levels of cellular visualization without the need for staining reagents and yields RNA with high RNA integrity numbers (&#8805;8) as needed for transcriptional profiling experiments.

This protocol uses a stain-free approach to visualize and isolate Purkinje cells in fresh-frozen tissue from human post-mortem cerebellum via laser capture microdissection. The purpose of this protocol is to generate sufficient amounts of high-quality RNA for RNA-sequencing.

Un protocole en acier inoxydable pour l’ARN haute qualité laser capturer des cellules de Microdissected Purkinje du cervelet humain Post-Mortem

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

Culture primaire des neurones isolés du cervelet de souris embryonnaire

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...