Muito obrigado a Elaine Pranski e Suwyn Carolyn para assistência técnica excelente e Harry LeVine III, Paul M. Murphy, e Marla Orientar para conversas importantes. O financiamento foi fornecido pela RR-00165, PO1AG026423, P50AG025688, AG030539, a Fundação Woodruff e da Emory University Research Committee.

Parte 1: Preparação dos homogeneizados de tecido esclarecidas <ol><li> Dounce homogeneizar o tecido cortical unfixed no volume 4 vezes de gelada tampão (0,1 M tampão fosfato [Ca + + - e Mg + + livre] mais 2x coquetel inibidor de protease [Santa Cruz]) com aproximadamente 30 golpes mesmo pilão (ie tampão adicionar 400μl a 100 mg de tecido). </li><li> Homogeneizados rodada por 5 minutos a 3.000 g (4 ° C). Remova cuidadosamente sobrenadante clarificado e alíquotas loja de extractos a -80 ° C até o uso. </li><li> Determinar a concentração de proteína total (mg / mL) para homogenatos esclareceu usando um ácido bicinchoninic (BCA) de ensaio, de acordo com as instruções do fabricante (ThermoFisher). </li></ol> Parte 2: SDS-PAGE Preparação da amostra <ol><li> Com um 10 bem, 10-20% tricine gel (Invitrogen), o volume máximo que pode ser carregado por poço é de aproximadamente 25μl. Volume total inclui tampão de amostra 2x SDS e agente redutor de 10x, de modo que o volume máximo de homogenato esclareceu que pode ser carregado por bem é 10μl. Esclareceu 20% (w / v) homogeneizados cortical deve ter uma concentração de proteína total superior a 5 mg / mL, permitindo a proteína total 50μg por poço. Amostras com menos de 5mg/ml proteína exigirá carregar menos proteína total por bem. No entanto, os níveis de proteína são ideais para a detecção de Aâ monomérica e agregados (50-60μg de proteína total é o ideal). Sempre carregue a mesma quantidade de proteína total por poço. </li><li> Para cada gel, carregar pelo menos um bem com 10μl de um marcador de peso molecular, tais como SeeBlue Plus2 (Invitrogen). Como um controle positivo, corrida de 10 100ng de Aβ40 sintéticos ou Aβ42 peptídeo (diluído em 1xPBS) em outro poço. </li><li> Prepare misturas de reação sobre o gelo, usando recém-descongelado, homogeneizados esclarecidas. Tubos de vórtice por 5-10 segundos, o calor em banho seco a 100 ° C por 5 minutos, em seguida, gire rapidamente todas as amostras para remover a condensação na tampa. </li></ol> Parte 3: SDS-PAGE de eletroforese em gel <ol><li> Carregar amostras agitadas em um gel tricine 10-20% na Gelbox XCell Bloqueio Celular Mini-Sure, usando tricine tampão SDS execução, de acordo com as instruções do fabricante (Invitrogen), </li><li> Executar gel a uma tensão constante de 125V por cerca de 90 minutos. Vamos executar amostras até que a banda marcador 4KDa é de cerca de 1 cm do fundo do gel. </li></ol> Parte 4: Proteínas de Transferência Géis para Membranas <ol><li> Remova cuidadosamente géis de caixa de plástico e montar o sanduíche de transferência dentro do Módulo II XCell Blot, de acordo com as instruções do fabricante (Invitrogen). Pré-molhada blotting almofadas e membranas de nitrocelulose com tampão de transferência de 0,2 Hm Tris-Glicina (20% metanol), e retire as bolhas de almofadas blotting ou o sanduíche de papel / filtro de membrana. </li><li> No Gelbox XCell, encha a câmara interna com tampão de transferência e encha a câmara externa com DIH 2 O (exposição ao metanol pode desgastar o plástico gelbox ao longo do tempo). </li><li> Executar a transferência por 2-3 horas em uma amperagem constante de 25mA. </li><li> Quando a transferência estiver completa, desconstruir o sanduíche e coloque o gel em DIH 2 O e as membranas em 1xPBS, tanto em pratos de plástico quadrado petri (ou outro recipiente adequado). </li><li> Para visualizar a eficiência da transferência de proteína, gel pode ser marcadas com SafeStain Simply Blue (Invitrogen), e as membranas com mancha vermelha Ponceau S (Sigma Aldrich), ambos de acordo com as instruções do fabricante. Esta coloração não irá interferir com immunoblotting. Se a coloração revela ineficiente transferência de proteína, modificar o tempo de transferência na Etapa 3. </li></ol> Parte 5. Antígeno-epítopo de recuperação e Immunoblotting <ol><li> Recuperação antigênica é um passo importante para revelar a epítopos Aâ na membrana para a ligação de anticorpos durante immunoblotting. Qualquer vapor, microondas ou banho de água que mantém uma temperatura constante de 100 ° C será suficiente. Para recuperação antigênica em um navio a vapor, calor selar a membrana em uma bolsa de plástico resistente Kapak preenchido com 1xPBS, à temperatura ambiente. Lay a bolsa apartamento em um navio a vapor pré-aquecido, uma vez que a bolsa começa a se expandir, incubar por mais 15 minutos. Permitir que a membrana para esfriar lentamente antes de removê-lo da bolsa, para evitar enrugamento excessivo. </li><li> À temperatura ambiente, remova cuidadosamente a membrana da bolsa vapor e enxaguar a membrana por 5 minutos em 1xPBS, seguido de um enxágüe de 5 minutos em TBS-T (Tris-Buffered pH, solução salina 8,0 com 0,05% Tween-20 [Sigma] ), em um agitador orbital. Incubar a membrana em solução de bloqueio (leite desnatado 2,5% em TBS-T) por uma hora, agitando. Sem enxaguar, transferência de membrana para um prato ou bolsa de plástico contendo o anticorpo primário diluído em solução de bloqueio (6E10 em 1:1.000 [1μg/ml] 1:5.000 [0.2μg/ml] diluição, Covance) bolha, evitandos. Incubar no agitador orbital em temperatura ambiente por uma hora e depois de 24-48 horas sacudindo a 4 ° C (mais de incubação pode dar um melhor sinal). </li><li> Lavar a membrana por 30 minutos em TBS-T (uma rápida lavagem seguido por 3 x 10 lavagens minuto). </li><li> Incubar a membrana no HRP-conjugado anticorpo secundário (Amersham ECL ovelhas anti-mouse, GE Healthcare), diluído 1:10000 em solução de bloqueio, por 90 minutos no agitador à temperatura ambiente. </li><li> Lavar a membrana por 30 minutos em TBS-T (uma rápida lavagem seguido por 3 x 10 lavagens minuto). </li><li> No agitador, incubar a membrana em recém-made SuperSignal reagente eletroquimioluminescência Pico West (ThermoFisher) por 5 minutos, limpe o excesso de reagente em papel que não solte fiapos do filtro e colocar a membrana em um cassete de filme, entre protetores da folha de plástico. Seque o reagente SuperSignal adicionais com poeira Kimwipes se necessário. </li><li> Expor a Kodak filme MR Biomax para intervalos de 30 segundos até 30 minutos e se desenvolver em um desenvolvedor de filme. Após 5 minutos, as faixas de monômero Aâ provavelmente será saturada. </li><li> Membranas pode ser despojado (após lavagem em TBS-T) por agitação em Restore Plus stripping tampão por 30 minutos em temperatura ambiente e reprobed com anticorpos adicionais se desejar. </li></ol> Parte 6: Representante Immunoblot: <img src="/files/ftp_upload/1916/1916fig1a.jpg" alt="Figura 1a" /> <img src="/files/ftp_upload/1916/1916fig1b.jpg" alt="Figura 1b" /> <img src="/files/ftp_upload/1916/1916fig1c.jpg" alt="Figura 1c" /> Figuras 1a-c. Homogeneizados Esclarecido contendo 50μg de proteína total de 7 seres humanos são analisados ​​para a presença de Aâ multimérica e APP. Immunoblotting com anticorpo 6E10 revela Aâ monômeros, dímeros, trímeros, tetrâmeros e APP (faixa superior) em todos os casos de Alzheimer, bem como abundantes maior multímeros de peso molecular Aâ em 2 casos AD. Sintéticos Aβ40 confirma a identidade do menor bandas de peso molecular. AD: doença de Alzheimer, DLB: demência com corpos de Lewy, ND: Nondemented humano (a) exposição do filme de 30 minutos, (b) a exposição de filmes de 5 minutos, (c) 30 a exposição segundo filme..

<ol>
	<li>Hardy, J., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+hypothesis+of+Alzheimer's+disease:+progress+and+problems+on+the+road+to+therapeutics.">The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics.</a> Science. 297, 353-356 (2002).</li><li>Haass, C., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+protein+oligomers+in+neurodegeneration:+lessons+from+the+Alzheimer's+amyloid+beta-peptide.">Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide.</a> Nat. Rev. Mol. Cell. Biol. 8, 101-112 (2007).</li><li>Walker, L. C., LeVine, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cerebral+proteopathies.">The cerebral proteopathies.</a> Neurobiol. Aging. 21, 559-561 (2000).</li><li>Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging,+amyloid,+and+Alzheimer's+disease:+a+perspective+in+honor.">Aging, amyloid, and Alzheimer's disease: a perspective in honor.</a> of Carl Cotman. Neurochem. Res. 28, 1705-1713 (2003).</li><li>Walsh, D. M., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abeta+oligomers+-+a+decade+of+discovery.">Abeta oligomers - a decade of discovery.</a> J. Neurochem. 101, 1172-1184 (2007).</li><li>Swerdlow, P. S., Finley, D., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+immunoblot+sensitivity+by+heating+of+hydrated+filters.">Enhancement of immunoblot sensitivity by heating of hydrated filters.</a> Anal. Biochem. 156, 147-153 (1986).</li><li>Ida, N., Hartmann, T., Pantel, J., Schr&#246;der, J., Zerfass, R., F&#246;rstl, H., Sandbrink, R., Masters, C. L., Beyreuther, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+hetergeneous+A4+peptides+in+human+cerebrospinal+fuid+and+blood+by+a+newly+developed+sensitive+Western+blot+assay.">Analysis of hetergeneous A4 peptides in human cerebrospinal fuid and blood by a newly developed sensitive Western blot assay.</a> J. Biol. Chem. 271, 22908-22914 (1996).</li></ol>

Apesar da importância da agregação Aâ na patogênese da doença de Alzheimer 1,4,5, poucos estudos têm descrito ou quantificado a distribuição de diversos multímeros Aâ humana em amostras de tecido cortical 2. Comumente usado técnicas de imunohistoquímica não permitem a discriminação de distintas espécies Aâ multimérica no tecido cortical fixo. Em unfixed homogeneizados de tecido cortical, multímeros Aâ podem ser separados e bioquimicamente avaliada através de eletroforese em ge...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Complete Protease Inhibitor Cocktail Tablets</td>
<td>&nbsp;</td>
<td>Santa Cruz Biotechnology</td>
<td>Sc-29130</td>
<td>1 tablet in 25ml buffer</td>
</tr>
<tr>
<td>BCA Protein Assay kit</td>
<td>&nbsp;</td>
<td>ThermoFisher Scientific</td>
<td>23225</td>
<td>&nbsp;</td>
<tr>
<td>XCell SureLock Mini-Cell and XCell II Blot Module Kit CE Mark</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>EI0002</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine SDS Sample Buffer (2X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC1676</td>
<td>&nbsp;</td>
<tr>
<td>NuPAGE Sample Reducing Agent (10X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>NP0004</td>
<td>&nbsp;</td>
<tr>
<td>SeeBlue Plus2 Pre-Stained Standard</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC5925</td>
<td>&nbsp;</td>
<tr>
<td>Novex 10-20% Tricine Gel 1.0 mm, 10 well</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>EC6625BOX</td>
<td>&nbsp;</td>
<tr>
<td>Nitrocellulose membranes, 0.2 &mu;m pore size</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC2000</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine SDS Running Buffer (10X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC1675</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tris-Glycine Transfer Buffer (25X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC3675</td>
<td>&nbsp;</td>
<tr>
<td>SimplyBlue SafeStain</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC6060</td>
<td>Will not interfere with immunostaining</td>
</tr>
<tr>
<td>ATX Ponceau S Red staining solution</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>09276</td>
<td>Will not interfere with immunostaining</td>
</tr>
<tr>
<td>Kapak heat sealable plastic sample pouches</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>0181225AA</td>
<td>&nbsp;</td>
<tr>
<td>6E10 mouse monoclonal antibody to A&beta;(1-16)</td>
<td>&nbsp;</td>
<td>Covance</td>
<td>SIG-39320</td>
<td>Dilute 1:1,000 up to 1:5,000 for WB</td>
</tr>
<tr>
<td>Tween 20</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>P2287</td>
<td>&nbsp;</td>
<tr>
<td>ECL Mouse IgG, HRP-Linked Whole A&beta; (from sheep)</td>
<td>&nbsp;</td>
<td>GE Healthcare Life Sciences</td>
<td>NA931-1ML</td>
<td>Dilute at 1:10,000</td>
</tr>
<tr>
<td>SuperSignal West Pico Chemiluminescent Substrate</td>
<td>&nbsp;</td>
<td>ThermoFisher</td>
<td>34077</td>
<td>&nbsp;</td>
<tr>
<td>Kodak Biomax MR Film</td>
<td>&nbsp;</td>
<td>Carestream Health</td>
<td>870 1302</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

sds-page/immunoblot detection of a&beta; multimers in human cortical tissue homogenates using antigen-epitope retrieval

O dobramento anômala e polimerização da β-amilóide (Aâ) peptídeo é pensado para iniciar a cascata neurodegenerativa na patogênese da doença de Alzheimer<sup&gt; 1</sup&gt;. Aâ é predominantemente um 40 - ou 42 peptídeo de ácido-amino que está propenso a auto-agregação em β folhas ricas em fibrilas amilóides que são encontrados nos núcleos das placas senis cerebral na doença de Alzheimer. A evidência crescente sugere que o baixo peso molecular, multímeros Aâ solúveis são mais tóxicos que fibrilar amilóide Aâ<sup&gt; 2</sup&gt;. A identificação e quantificação de baixo e alto peso molecular de espécies Aâ multimérica no tecido cerebral é um objectivo essencial na pesquisa da doença de Alzheimer, e os métodos utilizados também podem ser aplicados para a identificação e caracterização de multímeros tóxicos em outras proteopathies<sup&gt; 3</sup&gt;. Naturalmente multímeros Aâ pode ser detectada por SDS-eletroforese em gel de poliacrilamida seguido por immunoblotting com Aâ anticorpos específicos. No entanto, a separação e detecção de Aâ multimérica requer o uso de altamente concentrado homogeneizados cortical e recuperação antigênica em pequenas membranas de poros do tamanho de nitrocelulose. Aqui nós descrevemos uma técnica para a preparação de esclarecer humanos homogeneizados cortical, a separação de proteínas por SDS-PAGE, e antígeno-epítopo de recuperação / Western blotting com anticorpo 6E10 para a região N-terminal do peptídeo Aâ. Usando este protocolo, que consistentemente detectar Aâ monômeros, dímeros, trímeros, tetrâmeros, e multímeros de peso molecular maior no tecido cortical dos seres humanos com a patologia de Alzheimer.

Watch this Scientific Journal Video about SDS-PAGE/Immunoblot Detection of AÎ² Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval at JoVE.com

SDS-PAGE/Immunoblot Detection of A&amp;#946; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

Nós descrevemos uma técnica para a preparação de esclarecer humanos homogeneizados cortical, separação de proteínas por SDS-PAGE, recuperação antigênica e immunoblotting com um anticorpo para o peptídeo Aâ. Usando este protocolo, que consistentemente detectar Aâ monomérica e multimérica no tecido cortical dos seres humanos com a patologia de Alzheimer.

SDS-PAGE/Immunoblot Detecção de multímeros Aâ no tecido cortical Humanos homogeneizados utilizando antígeno Epitope-Retrieval

The anomalous folding and polymerization of the &beta;-amyloid (A&beta;) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's ...

sds-pageimmunoblot-detection-multimers-human-cortical-tissue

Research

JoVE Journal

Neuroscience

SDS-PAGE/Immunoblot Detection of A&beta; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

24.0K visualizações.  Emory University. Nós descrevemos uma técnica para a preparação de esclarecer humanos homogeneizados cortical, separação de proteínas por SDS-PAGE, recuperação antigênica e immunoblotting com um anticorpo para o peptídeo Aâ. Usando este protocolo, que consistentemente detectar Aâ monomérica e multimérica no tecido cortical dos seres humanos com a patologia de Alzheimer.

Vídeo: SDS-PAGE/Immunoblot Detection of A&#946; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1&deg;C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Criopreservação de tecido cortical Blocos de Geração de culturas altamente enriquecido Neuronal

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

Neurociência

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Lapso de tempo de imagens ao vivo de relação clonal células progenitoras neurais no desenvolvimento Zebrafish Encéfalo anterior

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research. Mammalian DNA is enzymatically modified at the 5th carbon position of cytosine (C) residues to 5-mC, predominately in the context of CpG dinucleotides. 5-mC is amenable to enzymatic oxidation to 5-hmC by the Tet family of enzymes, which are believed to be involved in development and disease. Currently, the biological role of 5-hmC is not fully understood, but is generating a lot of interest due to its potential as a biomarker. This is due to several groundbreaking studies identifying 5-hydroxymethylcytosine in mouse embryonic stem (ES) and neuronal cells. Research techniques, including bisulfite sequencing methods, are unable to easily distinguish between 5-mC and 5-hmC . A few protocols exist that can measure global amounts of 5-hydroxymethylcytosine in the genome, including liquid chromatography coupled with mass spectrometry analysis or thin layer chromatography of single nucleosides digested from genomic DNA. Antibodies that target 5-hydroxymethylcytosine also exist, which can be used for dot blot analysis, immunofluorescence, or precipitation of hydroxymethylated DNA, but these antibodies do not have single base resolution.In addition, resolution depends on the size of the immunoprecipitated DNA and for microarray experiments, depends on probe design. Since it is unknown exactly where 5-hydroxymethylcytosine exists in the genome or its role in epigenetic regulation, new techniques are required that can identify locus specific hydroxymethylation. The EpiMark 5-hmC and 5-mC Analysis Kit provides a solution for distinguishing between these two modifications at specific loci. The EpiMark 5-hmC and 5-mC Analysis Kit is a simple and robust method for the identification and quantitation of 5-methylcytosine and 5-hydroxymethylcytosine within a specific DNA locus. This enzymatic approach utilizes the differential methylation sensitivity of the isoschizomers MspI and HpaII in a simple 3-step protocol. Genomic DNA of interest is treated with T4-BGT, adding a glucose moeity to 5-hydroxymethylcytosine. This reaction is sequence-independent, therefore all 5-hmC will be glucosylated; unmodified or 5-mC containing DNA will not be affected. This glucosylation is then followed by restriction endonuclease digestion. MspI and HpaII recognize the same sequence (CCGG) but are sensitive to different methylation states. HpaII cleaves only a completely unmodified site: any modification (5-mC, 5-hmC or 5-ghmC) at either cytosine blocks cleavage. MspI recognizes and cleaves 5-mC and 5-hmC, but not 5-ghmC. The third part of the protocol is interrogation of the locus by PCR. As little as 20 ng of input DNA can be used. Amplification of the experimental (glucosylated and digested) and control (mock glucosylated and digested) target DNA with primers flanking a CCGG site of interest (100-200 bp) is performed. If the CpG site contains 5-hydroxymethylcytosine, a band is detected after glucosylation and digestion, but not in the non-glucosylated control reaction. Real time PCR will give an approximation of how much hydroxymethylcytosine is in this particular site. In this experiment, we will analyze the 5-hydroxymethylcytosine amount in a mouse Babl/C brain sample by end point PCR.

The EpiMark 5-hmC and 5-mC Analysis Kit can be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a spe cific locus. The kit distinguishes 5-mC from 5-hmC by the addition of glucose to the hydroxyl group of 5-hmC via an enzymatic reaction utilizing &beta;-glucosyltransferase (T4-BGT). When 5-hmC occurs In the context of CCGG, this modification converts a cleavable MspI site to a non-cleavable site.

Alta Sensibilidade de Detecção de 5 hydroxymethylcytosine-in Balb / C Tecido Cerebral

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research.   Mammalian DNA is enzymatically ...

Coherence between Brain Cortical Function and Neurocognitive Performance during Changed Gravity Conditions

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and focused on psychological aspects. Until now, objective observation of neurophysiological parameters has not been carried out - undoubtedly because the technical and methodological means have not been available -, investigations into the neurophysiological effects of weightlessness are in their infancy (Schneider et al. 2008).
While imaging techniques such as positron emission tomography (PET) and magnetic resonance imaging (MRI) would be hardly applicable in space, the non-invasive near-infrared spectroscopy (NIRS) technique represents a method of mapping hemodynamic processes in the brain in real time that is both relatively inexpensive and that can be employed even under extreme conditions. The combination with electroencephalography (EEG) opens up the possibility of following the electrocortical processes under changing gravity conditions with a finer temporal resolution as well as with deeper localization, for instance with electrotomography (LORETA).
Previous studies showed an increase of beta frequency activity under normal gravity conditions and a decrease under weightlessness conditions during a parabolic flight (Schneider et al. 2008a+b). Tilt studies revealed different changes in brain function, which let suggest, that changes in parabolic flight might reflect emotional processes rather than hemodynamic changes. However, it is still unclear whether these are effects of changed gravity or hemodynamic changes within the brain. Combining EEG/LORETA and NIRS should for the first time make it possible to map the effect of weightlessness and reduced gravity on both hemodynamic and electrophysiological processes in the brain. Initially, this is to be done as part of a feasibility study during a parabolic flight. Afterwards, it is also planned to use both techniques during medium- and long-term space flight.
It can be assumed that the long-term redistribution of the blood volume and the associated increase in the supply of oxygen to the brain will lead to changes in the central nervous system that are also responsible for anaemic processes, and which can in turn reduce performance (De Santo et al. 2005), which means that they could be crucial for the success and safety of a mission (Genik et al. 2005, Ellis 2000).
Depending on these results, it will be necessary to develop and employ extensive countermeasures. Initial results for the MARS500 study suggest that, in addition to their significance in the context of the cardiovascular and locomotor systems, sport and physical activity can play a part in improving neurocognitive parameters. Before this can be fully established, however, it seems necessary to learn more about the influence of changing gravity conditions on neurophysiological processes and associated neurocognitive impairment.

The effect of weightlessness and hypergravity on both hemodynamic and electrophysiological processes in the brain is going to be followed during parabolic flight by EEG and NIRS techniques. 
A feasibility study of a more complex experiment, which is planned to carry out during medium- and long-term space flight.

Coerência entre a função cerebral cortical e Desempenho neurocognitiva durante condições de gravidade mudou

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and ...

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Geração de células-tronco neurais de Discarded tecido fetal humano Cortical

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

Imagem de cálcio funcional em desenvolvimento de redes Cortical

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

Quantificação de filamentosas actina (F-actina) puncta no Rat neurônios corticais

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

Quantificação de Endosome e Moosidades de Lisosoma em Neurônios Cultivados Usando Sondas Fluorescentes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Visualization of Cortical Modules in Flattened Mammalian Cortices

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and can be delineated by certain histochemical and immunohistochemical methods. In this study, we highlight a method to isolate the cortex from mammalian brains and flatten them to obtain sections parallel to the cortical sheet. We further highlight selected histochemical and immunohistochemical methods to process these flattened tangential sections to visualize cortical modules. In the somatosensory cortex of various mammals, we perform cytochrome oxidase histochemistry to reveal body maps or cortical modules representing different parts of the body of the animal. In the medial entorhinal cortex, an area where grid cells are generated, we utilize immunohistochemical methods to highlight modules of genetically determined neurons which are arranged in a grid-pattern in the cortical sheet across several species. Overall, we provide a framework to isolate and prepare layer-wise flattened cortical sections, and visualize cortical modules using histochemical and immunohistochemical methods in a wide variety of mammalian brains.

This article describes a detailed methodology to obtain flattened tangential sections from mammalian cortices and visualize cortical modules using histochemical and immunohistochemical methods.

Visualização de módulos corticais em córtices mamíferos achatadas

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and ...

Statistical Modelling of Cortical Connectivity Using Non-invasive Electroencephalograms

Non-invasive electrophysiological recordings are useful for the evaluation of nervous system function. These techniques are inexpensive, fast, replicable, and less resource-intensive than imaging. Further, the functional data produced have excellent temporal resolution, which is not achievable with structural imaging.
Current applications of electroencephalograms (EEG) are limited by data processing methods. Standard analysis techniques using raw time series data at individual channels are very limited methods of interrogating nervous system activity. More detailed information about cortical function can be achieved by examining relationships between channels and deriving statistical models of how areas are interacting, allowing visualization of connectivity between networks.
This manuscript describes a method for deriving statistical models of cortical network activity by recording EEG in a standard manner, then examining the interelectrode coherence measures to assess relationships between the recorded areas. Higher order interactions can be further examined by assessing the covariance between the coherence pairs, producing high-dimensional &#34;maps&#34; of network interactions. These data constructs can be examined to assess cortical network function and its relationship to pathology in ways not achievable with traditional techniques.
This approach offers greater sensitivity to network level interactions than is achievable with raw time series analysis. It is, however, limited by the complexity of drawing specific mechanistic conclusions about the underlying neural populations and the high volumes of data generated, requiring more advanced statistical techniques for evaluation, including dimensionality reduction and classifier-based approaches.

Standard EEG analysis techniques offer limited insight into nervous system function. Deriving statistical models of cortical connectivity offers far greater ability to investigate underlying network dynamics. Improved functional assessment opens new possibilities for diagnosis, prognostication, and outcome prediction in nervous system diseases.

Modelagem estatística da conectividade cortical usando eletroencefalogramas não invasivos

Non-invasive electrophysiological recordings are useful for the evaluation of nervous system function. These techniques are inexpensive, fast, replicable, ...