Os autores gostariam de agradecer a James Oesterling para citometria de fluxo assistência. Esta pesquisa foi suportada por uma concessão do Instituto Nacional do Câncer (R01-CA178380, ZY) e uma bolsa de US National Institutes de Pós-Doutorado formação (5T32CA009035-37, Lwy).

A utilização de animais neste protocolo foi realizado de acordo com os procedimentos aprovados pelo Comitê Cancer Center, Animal Care and Use a Fox Chase. 1. preparação de instrumentos, Solutions e Lamelas <ol><li> Limpe dois conjuntos de # 5 pinça fina, um conjunto de # 7 pinças curvas finas, uma grande tesoura cirúrgica, uma tesoura microdissecting, uma espátula e uma colher perfurada. Coloque os instrumentos em uma auto-selante bolsa de esterilização e autoclave. Mantenha instrumentos em bolsa até que esteja pronto para uso. </li><li> Prepara-se uma solução de agarose de baixo ponto de fusão de 3%. <ol><li> Adicionar 1,5 g de agarose em 50 ml de tampão fosfato salina estéril por Dulbecco (DPBS) em seguida, coloque no microondas e aqueça até que apareçam bolhas. Frasco do redemoinho e reaquecimento até que todos agarose foi dissolvida. Misturar a solução com uma barra de agitação, antes do aquecimento, se aglomerados aparecer. </li><li> Solução da loja em um banho de água a 37 ° C até ser necessário. </li><li> Para armaze longo prazoe, armazenar a solução de agarose para uso futuro, à temperatura ambiente ou a 4 ° C, durante meses. Reaquecimento no microondas para liquefazer. Para o aquecimento repetido da solução de agarose, pesar o frasco antes de aquecimento para manter o seu peso inicial, com água destilada estéril quente. </li></ol></li><li> Preparar as seguintes soluções para a dissociação de células à base de papaína como descrito previamente 2: <ol><li> A solução de papaína com papaína (100 U / ml), cisteína (0,2 mg / ml) e ADNase (250 U / ml) em fenol vermelho estéril contendo DPBS. </li><li> Uma solução ovomucóide com albumina de soro bovino (BSA, 8 mg / ml), inibidor de tripsina de soja (8 mg / ml)) e DNase (250 U / ml) em fenol estéril contendo vermelho-DPBS. Filtrar esterilizar ambas as soluções e ajustar o pH para retornar cor original de fenol contendo vermelho-DPBS. </li></ol></li><li> Preparar meios de cultura de células (NB / B27): complementar meio basal com piruvato de sódio 1 mM, 2 mM de L-glutamina, 1% de penicilina-estreptomicina (P / S) e suplemento de B27. Filter esterilizar e proteger da luz. Equilibrar media a 37 ° C e 5% de CO 2 antes da sua utilização. </li><li> Preparar tampão de FACS: 5% de soro fetal bovino (FBS) em DPBS. Adicionar 2,5 ml de FBS a 47,5 ml de DPBS. </li><li> Prepare a poli-D-lisina (PDL) -Revestido lamelas. <ol><li> Autoclave novas lamelas de vidro. </li><li> Revestir as lamelas com PDL (100 ug / ml em água destilada estéril) e incubar durante 2 horas a 37 ° C ou à temperatura ambiente durante a noite. </li><li> Lavar lamelas com água destilada estéril. Lave uma vez com meio NB / B27. Vamos lamelas secar completamente no tecido de cultura capa antes de usar. </li></ol></li></ol> 2 Dissecção e Preparação de fatias de tecido <ol><li> No dia da dissecção, limpe a área de dissecção com etanol e 70% de cobertura com um pano absorvente. </li><li> Remover ferramentas de dissecção da bolsa de esterilização. Adicionar gelada, DPBS estéril / 1% P / S para uma pequena placa de Petri e colocar no gelo. </li><li> Decapitaria P4 Math1-GFP / nestin-PCP filhotes de rato com grandes tesouras cirúrgicas em seguida, mantenha a cabeça para baixo com uma pinça curva finas. Retire a pele e descascar delicadamente fora o crânio com # 5 pinça fina, em seguida retire o cérebro do crânio utilizando uma espátula e transfira-o para a placa de Petri contendo DPBS / 1% P / S. <ol><li> Usando # 5 fórceps finos, cuidadosamente separar o cerebelo a partir do resto do cérebro. Certifique-se de dissecar um filhote de cada vez mais rápido possível. </li><li> Recolha um mínimo de 8 cerebelo, a fim de obter NEPs suficientes para análises moleculares e funcionais. </li></ol></li><li> Encher um molde medindo a incorporação de 2 x 2 x 2 cm de altura com uma solução de agarose de baixo ponto de fusão de 3%. Certifique-se de que a temperatura de agarose é não mais do que 37 ° C. </li><li> Retire o excesso de líquido do cerebelo, esfregando suavemente sobre um tecido de laboratório. Coloque cerebelo em molde na posição vertical. Coloque o molde em gelo para permitir que solidifique rapidamente. Use ~ 4 cerebelo por bloco. </li><li> Obter licortes de tecido ving com uma lâmina vibratome vibrando. Apare contendo cerebelo bloco de agarose com uma lâmina de barbear. Cole o bloco de agarose para a placa com vibratome orientada na posição vertical, os cerebelos. </li><li> Encha a bandeja do vibratome com geladas DPBS estéril / 1% P / S e local de gelo embaixo. <ol><li> Cortar 600 uM secções através de todo o comprimento do cerebelo. Colete seções da bandeja de gelo utilizando uma colher perfurada. Seções coloque em um prato de Petri cheia de DPBS / 1% P / S no gelo. </li></ol></li></ol> 3. Microdissecção e dissociação celular <ol><li> Coloque a placa de Petri contendo fatias de tecido sob um microscópio de dissecação fluorescente. <ol><li> Cuidadosamente separar o EGL do resto da secção de cerebelar usando uma pinça fina dissecando entre o PCP + camada molecular e a GFP + EGL (referem-se a linha a tracejado na Figura 1). Ter cuidado para não incluir qualquer parte da camada molecular, o que irá resultar emcontaminação de expressando nestina Bergmann glia. </li><li> Conclua esta etapa, tão rapidamente quanto possível e manter o tecido em gelo para evitar a morte celular. </li><li> Retire a agarose em torno do tecido antes de prosseguir para a próxima etapa. </li></ol></li><li> Digerir o tecido EGL microdissecados utilizando um protocolo baseado em papaína como descrito previamente 2 para se obter uma suspensão de célula única. <ol><li> Re-suspender as células em tampão FACS para a coleta de células CFP-positivos através de FACS. </li></ol></li></ol> 4 células de triagem e Galvanização <ol><li> Ordenação de células de fluorescência utilizando um PCP e de alta velocidade de fluxo estéril citómetro contendo um filtro apropriado PCP e recolher células em tampão de SCAF em gelo. Obter aproximadamente 100.000 NEPs de cada cerebelo P4. </li><li> Células centrifugar a 300 xg por 5 min. Utilizar imediatamente células para análise molecular ou cultura para mais experimentação. </li><li> Para células de cultura, re-suspender as células pré-aquecido NB / B27 mEDIA e placa em lamelas PDL-revestidos como descrito anteriormente 2. </li></ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+neural+cell+fates+and+medulloblastoma+in+mouse+patched+mutants.">Altered neural cell fates and medulloblastoma in mouse patched mutants.</a> Science. 277, 1109-1113 (1997).</li><li>Li, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+population+of+Nestin-expressing+progenitors+in+the+cerebellum+exhibits+increased+tumorigenicity.">A population of Nestin-expressing progenitors in the cerebellum exhibits increased tumorigenicity.</a> Nat Neurosci. 16, 1737-1744 (2013).</li><li>Ben-Arie, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Math1+is+essential+for+genesis+of+cerebellar+granule+neurons.">Math1 is essential for genesis of cerebellar granule neurons.</a> Nature. 390, 169-172 (1997).</li><li>Sutter, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+stem+cells+act+as+medulloblastoma-initiating+cells+in+a+mouse+model+and+a+neural+stem+cell+signature+characterizes+a+subset+of+human+medulloblastomas.">Cerebellar stem cells act as medulloblastoma-initiating cells in a mouse model and a neural stem cell signature characterizes a subset of human medulloblastomas.</a> Oncogene. 29, 1845-1856 (2010).</li><li>Sotelo, C., Alvarado-Mallart, R. M., Frain, M., Vernet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+plasticity+of+adult+Bergmann+fibers+is+associated+with+radial+migration+of+grafted+Purkinje+cells.">Molecular plasticity of adult Bergmann fibers is associated with radial migration of grafted Purkinje cells.</a> J Neurosci. 14, 124-133 (1994).</li><li>Lumpkin, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Math1-driven+GFP+expression+in+the+developing+nervous+system+of+transgenic+mice.">Math1-driven GFP expression in the developing nervous system of transgenic mice.</a> Gene Expr Patterns. 3, 389-395 (2003).</li><li>Encinas, J. 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O método descrito aqui é microdissecção o primeiro procedimento capaz de isolar uma população pura de células vivas para utilização em análises moleculares e funcionais. Como demonstrado por Li et al. 9, NEPs obtidos por este método podem ser cultivadas e retomado em diferentes experiências e por causa do seu elevado grau de pureza, pode também ser utilizado para a análise de microarray. É muito importante ter tempo para se tornar proficientes co...

O cerebelo é composta de várias camadas de células, cada uma contendo tipos celulares distintas. Durante o desenvolvimento, a EGL contém proliferando grânulos dos neurônios precursores (PNB), enquanto a camada molecular e camada de Purkinje conter Bergmann glia e neurônios de Purkinje, respectivamente. Profundamente dentro do cerebelo encontra-se a substância branca, que contém células-tronco neurais (NCCC) e oligodendrócitos 1. Sonic hedgehog (Shh) desempenha um papel fundamental na regulação do desenvolvimento do cerebelo e, em particular, que promove a proliferação de PNB através da ligação ao seu receptor de Patched (Ptc), um regulador negativo da sinalização Shh 2-4. A ativação anormal de sinalização Shh gera meduloblastoma (MB), o tumor cerebral maligno mais comum em crianças 5,6. PTC MB mutantes modelos transgênicos são ferramentas poderosas para o estudo da MB e desenvolver tumores que lembram MB humano 7,8. Usando estesratos, foi descoberto que PNB são a célula de origem para hedgehog de ​​tipo MB 7. Além disso, em adição ao PNB, que recentemente identificada uma população única de células progenitoras neuronais dentro da EGL do cerebelo em desenvolvimento, que também pode dar origem a MB. Estas células expressam níveis elevados da proteína de tipo VI filamento intermediário, nestina, e são denominados NEPs 9. NEPs estão localizados na parte profunda da EGL existir transitoriamente e apenas durante o desenvolvimento do cerebelo neonatal. Eles estão comprometidos com a linhagem de células granulares como PNB, mas são distintos como eles estão quietos e não expressam Math1, um marcador bem estabelecido para PNB convencionais 10. Além disso, NEPs dar origem a MB de forma mais eficiente do que o PNB após a ativação da via de sinalização Shh 9, que lhes uma nova origem para MB tumorigenesis faz. Nós NEPs da EGL cerebelar previamente isolada no dia pós-natal 4 (P4) pela técnica de microdissecçãodescrito aqui 9. Microdissecção visualização microscópica directa através do tecido permite a dissecção específica da EGL cerebelar. Isto é necessário porque Nestin também é expressa por NSC na substância branca do cerebelo e por Bergmann glia na camada molecular 1,7,11,12 e era crucial que estas células não ser incluídos, como eles iriam confundir análise. As células isoladas a partir de tecido microdissecados pode ser imediatamente utilizado para a análise molecular, ou podem ser cultivadas para outras aplicações. Para ajudar na especificamente microdissecting a EGL e para purificar ainda mais NEPs, ratos Math1-GFP (proteína fluorescente verde) foram cruzados com camundongos Nestin-CFP (proteína fluorescente ciano). O factor de transcrição de Math1 é especificamente expresso por PNB e expressão da GFP é claramente visível na EGL 9,13. Ratinhos nestina-PCP expressar uma forma nuclear de PCP e podem ser facilmente visualizadas na camada molecular 9,14. Juntos, GFP e expressão PCP criar limites para microdissecção do EGL (ver Figura 1). NEPs PCP-positivos foram então isolados por FACS, após dissociação enzimática das GECV dissecados. Actualmente, o método mais bem utilizados para isolar células da EGL é centrifugação em gradiente de Percoll de tecido cerebelar toda 15,16. Este método, no entanto, não é capaz de excluir completamente populações celulares nestina + a partir da camada molecular e da matéria branca e, portanto, não pode ser usado para o estudo de NEPs. Microdissecção captura a laser, que utiliza a transferência de energia laser para remover as células de interesse, é outro método usado para isolar células específicas dentro de um tecido heterogéneo 17,18 mas apenas células capturadas podem ser usadas para a recuperação de ADN, ARN e proteína e não está capazes de ser cultivadas. Este protocolo fornece uma maneira de isolar especificamente um puro população vivo, de NEPs da EGL.Esta técnica também pode ser aplicado para diferentes tipos de tecidos que tenham ou arquitectura anatómica reconhecível ou marcados com fluorescência células / regiões. Por conseguinte, a principal vantagem de incorporar esta técnica microdissecção novo em protocolos de isolamento de células é que as células podem ser isoladas a partir de regiões específicas do tecido para eliminar os contaminantes de tecidos vizinhos e as células podem ser recolhidas para análise imediatamente ou cultivadas durante outras aplicações.

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			<td height="42" style="height:42px;width:215px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
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			<td style="width:167px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:215px;">Neurobasal media</td>
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			<td height="21" style="height:21px;width:215px;">PDL</td>
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			<td height="42" style="height:42px;width:215px;">ultra pure low melting temperature agarose</td>
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isolation of distinct cell populations from the developing cerebellum by microdissection

Microdissecção é uma nova técnica que pode isolar regiões específicas de um tecido e eliminar a contaminação a partir de fontes celulares em áreas adjacentes. Este método foi utilizado pela primeira vez no estudo de nestina expressa progenitoras (NEPs), uma população de células recentemente identificados na camada germinal externa cerebelar (EGL). Usando microdissecção em combinação com separação de células activada por fluorescência (FACS), uma população pura de NEPs foi recolhido separadamente, a partir de precursores neuronais dos grânulos convencionais na EGL e de outros contaminantes células que expressam nestina-no cerebelo. Sem microdissecção, análises funcionais de NEPs não teria sido possível com os atuais métodos disponíveis, tais como a centrifugação em gradiente de Percoll e captura a laser microdissecção. Esta técnica também pode ser aplicado para utilização com vários tecidos que contêm tanto regiões conhecidas ou células marcadas fluorescentemente. Mais importante ainda, uma grande vantagem do presente microdissection técnica é que as células isoladas são de vida e pode ser cultivado para experimentação posterior, o que não é actualmente possível com os outros métodos descritos.

Como mostrado na Figura 1A, as fatias de cerebelo foram preparadas a partir de P4 Math1-GFP / mice nestina-PCP, em que a EGL cerebelar foram dominados por PNB-expressando GFP e a camada molecular foi enriquecida por células gliais PCP-positivos. Para isolar NEPs que estão localizadas na parte profunda da EGL, fatias foram microdissecadas entre o EGL e a camada molecular (ao longo da linha tracejada, Figura 1A). GECV dissecado (Figura 1B) foram, em se...

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

Progenitores que expressam nestina são uma população recentemente identificada de progenitores neuronais no cerebelo em desenvolvimento. Usando a técnica de microdissecção aqui apresentados em combinação com separação de células activada por fluorescência, esta população de células pode ser purificado sem contaminação de outras regiões do cerebelo e podem ser cultivadas para estudos posteriores.

O isolamento de populações de células distintas do cerebelo em desenvolvimento por Microdissecção

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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Neuroscience

9.5K visualizações.  Temple University Health System.  Progenitores que expressam nestina são uma população recentemente identificada de progenitores neuronais no cerebelo em desenvolvimento. Usando a técnica de microdissecção aqui apresentados em combinação com separação de células activada por fluorescência, esta população de células pode ser purificado sem contaminação de outras regiões do cerebelo e podem ser cultivadas para estudos posteriores. 

Vídeo: 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.

Caracterização eletrofisiológica de GFP-Expressando populações de células na retina intacta

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

Neurociência

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.

Laser Microdissecção Captura de Populações enriquecida de neurônios ou neurônios individuais para análise de expressão gênica após a lesão cerebral traumática

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.

Produção de RNA para análise transcriômica de corpos celulares de neurônio motor da medula espinhal do rato por microdisseção de captura a 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.

Imagens ao vivo da mitose no desenvolvimento Cortex embrionárias de camundongos

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.

Captura Laser Microdissection - uma demonstração do Isolamento de Pessoa dopamina Neurônios e região ventral tegmental Entire

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

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

Análise em profundidade Physiological de populações de células definido no aguda Tissue Fatias do mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

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.

Viver a imagem, seguida por uma única célula de rastreamento para biologia celular de Monitor e a progressão da linhagem de várias populações neurais

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.

Otimização de captura Laser Microdissection para o isolamento dos gânglios entéricos de tecido humano fresco congelado

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.

Um protocolo de aço inoxidável para isolamento de RNA de alta qualidade do Laser capturar células de Microdissected Purkinje no cerebelo humano 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.

Cultura primária de neurônios isolados do cerebelo embrionário do rato

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 ...