Molte grazie a Elaine Pranski e Carolyn Suwyn per un&#39;eccellente assistenza tecnica e di Harry LeVine III, M. Paul Murphy, e ingranaggi per le conversazioni importanti Marla. Il finanziamento è stato fornito da RR-00165, PO1AG026423, P50AG025688, AG030539, la Fondazione Woodruff e la Emory University Comitato di ricerca.

Parte 1: Preparazione di omogenati di tessuti chiarito <ol><li> Dounce omogeneizzare non fissati tessuto corticale in 4 volumi tempi del freddo buffer (0.1M PBS [Ca + + - e Mg + +-free] da cocktail più di proteasi 2x inibitore [Santa Cruz]) con circa 30 colpi, anche pestello (cioè tampone aggiungere 400μl a 100 mg di tessuto). </li><li> Omogenati Spin per 5 minuti a 3.000 g (4 ° C). Rimuovere con cautela surnatanti chiarito e aliquote vendite di questi estratti a -80 ° C fino al momento dell&#39;uso. </li><li> Determinare la concentrazione totale di proteine ​​(mg / mL) per omogenati chiarita utilizzando un acido bicinchoninic (BCA) test, secondo le istruzioni del produttore (ThermoFisher). </li></ol> Parte 2: SDS-PAGE La preparazione del campione <ol><li> Con un 10 e, il 10-20% Tricine gel (Invitrogen), il volume massimo che può essere caricato per bene è ~ 25μl. Volume totale comprende 2x tampone campione SDS e 10x agente riducente, in modo che il volume massimo di omogenato chiarito che possono essere caricati per bene è 10μl. Chiarito il 20% (w / v) omogenati corticali dovrebbe avere una concentrazione totale di proteine ​​superiore a 5 mg / mL, permettendo 50μg di proteine ​​totali per bene. I campioni con meno di 5mg/ml proteina richiederà meno carico di proteine ​​totali per bene. Tuttavia, i livelli di proteine ​​sono ottimali per la rilevazione di Aβ monomerica e aggregati (50-60μg delle proteine ​​totali è ottimale). Caricare sempre la stessa quantità di proteine ​​totali per bene. </li><li> Per ogni gel, carico di almeno un bene con 10μl di un marcatore di peso molecolare come SeeBlue Plus2 (Invitrogen). Come controllo positivo, eseguire 10-100 ng di sintesi Aβ40 o Aβ42 peptide (diluito in 1xPBS) in un altro bene. </li><li> Preparare miscele di reazione su ghiaccio, con appena scongelati, omogenati chiarite. Tubi vortex per 5-10 secondi, il calore in un bagno secco a 100 ° C per 5 minuti, quindi girare velocemente tutti i campioni per eliminare condensa nel tappo. </li></ol> Parte 3: SDS-PAGE elettroforesi su gel <ol><li> Caricare i campioni in agitazione su un gel Tricine 10-20% nel Certo XCell Blocco Mini-Cell Gelbox, utilizzando Tricine tampone SDS in esecuzione, secondo le istruzioni del produttore (Invitrogen), </li><li> Eseguire gel ad una tensione costante di 125V per circa 90 minuti. Lasciate che i campioni fino al marcatore 4KDa la band è di circa 1 cm dal fondo del gel. </li></ol> Parte 4: Trasferimento di proteine ​​da gel a Membrane <ol><li> Rimuovere con cura dal gel in plastica e assemblare il panino trasferimento all&#39;interno del modulo XCell II Blot, secondo le istruzioni del produttore (Invitrogen). Pre-umido assorbente pastiglie e le membrane di nitrocellulosa 0.2μm con Tris-Glicina buffer di trasferimento (20% metanolo), ed eliminare eventuali bolle dal pad assorbente o il filtro di carta / membrana sandwich. </li><li> Nel Gelbox XCell, riempire la camera interna con buffer di trasferimento e riempire la camera esterna con DIH 2 O (esposizione metanolo può logorare la gelbox plastica nel tempo). </li><li> Eseguire il trasferimento per 2-3 ore a un amperaggio costante di 25mA. </li><li> Quando il trasferimento è stato completato, decostruire il panino e posizionare il gel in DIH 2 O e le membrane in 1xPBS, sia in piazza scatole Petri in plastica (o altro contenitore appropriato). </li><li> Per visualizzare l&#39;efficienza del trasferimento di proteine, gel può essere colorato con i Simply Blue SafeStain (Invitrogen), e le membrane con Ponceau S macchia rossa (Sigma Aldrich), sia secondo le istruzioni del produttore. Questa colorazione non interferirà con immunoblotting. Se la colorazione rivela inefficiente trasferimento di proteine, modificare il tempo di trasferimento al punto 3. </li></ol> Parte 5. Antigene-epitopo recupero e Immunoblotting <ol><li> Recupero Antigen è un passo importante nel rivelare il epitopi Aβ sulla membrana il legame degli anticorpi durante immunoblotting. Ogni bagno a vapore, forno a microonde, o l&#39;acqua che mantiene una temperatura costante di 100 ° C sarà sufficiente. Per il recupero antigene in un piroscafo, termosaldato la membrana in un robusto sacchetto di plastica riempito di Kapak 1xPBS, a temperatura ambiente. Posare il sacchetto appartamento in un pre-riscaldato a vapore, una volta che il sacchetto comincia ad espandersi, incubare per altri 15 minuti. Lasciare che la membrana si raffreddi prima di rimuoverla dal sacchetto, per evitare pieghe eccessive. </li><li> A temperatura ambiente, rimuovere con attenzione la membrana dal sacchetto fumante e risciacquare la membrana per 5 minuti in 1xPBS, seguito da 5 minuti a sciacquare in TBS-T (Tris-Buffered Saline, pH 8,0 con 0,05% di Tween-20 [sigma] ), su un agitatore orbitale. Incubare la membrana in una soluzione bloccante (latte scremato 2,5% in TBS-T) per un&#39;ora, agitando. Senza risciacquo, trasferimento a membrana su un piatto o sacchetto di plastica contenente l&#39;anticorpo primario diluito in soluzione bloccante (6E10 a 1:1000 [1μg/ml] 1:5.000 [0.2μg/ml] diluizione, Covance), bolla evitandos. Incubare su un agitatore orbitale a temperatura ambiente per un&#39;ora e poi 24-48 ore in agitazione a 4 ° C (più lungo di incubazione può dare un segnale migliore). </li><li> Sciacquare la membrana per 30 minuti in TBS-T (una risciacquata veloce seguita da 3 x 10 risciacqui minuto). </li><li> Incubare la membrana in HRP-coniugato anticorpo secondario (Amersham ECL di pecora anti-topo, GE Healthcare), diluito in soluzione 1:10.000 blocco, per 90 minuti mediante l&#39;agitatore a temperatura ambiente. </li><li> Sciacquare la membrana per 30 minuti in TBS-T (una risciacquata veloce seguita da 3 x 10 risciacqui minuto). </li><li> L&#39;agitatore, incubare la membrana in appena fatto reagente Electrochemiluminescence SuperSignal Pico West (ThermoFisher) per 5 minuti, fuori macchia reagente in eccesso sulla privo di carta da filtro e collocare la membrana in un film di cassetta, tra protettori foglio di plastica. Blot off ulteriori reagente SuperSignal con polvere Kimwipes se necessario. </li><li> Esporre ad una pellicola Kodak Biomax MR per intervalli di 30 secondi fino a 30 minuti e si sviluppano in uno sviluppatore film. Dopo 5 minuti, le bande di monomero Aβ sarà probabilmente saturo. </li><li> Membrane possono essere rimossi (dopo il risciacquo in TBS-T), agitando in Restore Plus stripping tampone per 30 minuti a temperatura ambiente e reprobed con anticorpi aggiuntivi se lo si desidera. </li></ol> Parte 6: Rappresentante 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" /> Figure 1a-c. Omogenati Chiarito contenente 50μg di proteine ​​totali da 7 soggetti umani vengono analizzati per la presenza di Aβ multimerica e APP. Immunoblotting con anticorpi 6E10 rivela Aβ monomeri, dimeri, trimeri, tetrameri, e APP (banda superiore) in tutti i casi di Alzheimer, così come più abbondante peso molecolare multimeri Aβ in 2 casi AD. Sintetico Aβ40 conferma l&#39;identità delle bande basso peso molecolare. AD: malattia di Alzheimer, DLB: demenza con corpi di Lewy, ND: umana dementi (a) esposizione film di 30 minuti, (b) l&#39;esposizione film di 5 minuti, (c) 30 esposizione secondo film..

<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>

Nonostante l&#39;importanza di aggregazione Aβ nella patogenesi della malattia di Alzheimer 1,4,5, pochi studi hanno descritto o quantificata la distribuzione di diversi multimeri Aβ in campioni di tessuto umano corticale 2. Comunemente usato tecniche immunoistochimiche non consentono la discriminazione di specie distinte Aβ multimerica nel fisso tessuto corticale. Non fissati in omogenati di tessuto corticale, multimeri Aβ possono essere separati e biochimicamente valutata mediante elettrofore...

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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

Il ripiegamento anomalo e polimerizzazione del β-amiloide (Aβ) peptide è pensato di avviare la cascata neurodegenerative nella patogenesi della malattia di Alzheimer<sup&gt; 1</sup&gt;. Aβ è prevalentemente un 40 - peptide acido o 42 aminoacidi che è incline a auto-aggregazione in β-sheet ricco di fibrille amiloidi che si trovano nei nuclei delle placche senili cerebrali nella malattia di Alzheimer. Crescente evidenza suggerisce che a basso peso molecolare, multimeri Aβ solubile sono più tossici amiloide Aβ fibrillare<sup&gt; 2</sup&gt;. L&#39;identificazione e la quantificazione a basso e ad alto peso molecolare, specie Aβ multimerica nel tessuto cerebrale è un obiettivo essenziale nella ricerca sulla malattia di Alzheimer, e dei metodi impiegati può essere applicata per l&#39;identificazione e la caratterizzazione dei multimeri tossici in altre proteopathies<sup&gt; 3</sup&gt;. Naturalmente multimeri Aβ può essere rilevato da SDS-poliacrilammide gel elettroforesi seguita da immunoblotting con anticorpi specifici anti-Aβ. Tuttavia, la separazione e la rilevazione di Aβ multimerica richiede l&#39;uso di alta concentrazione omogenati corticale e recupero dell&#39;antigene in piccoli pori di dimensioni membrane di nitrocellulosa. Qui si descrive una tecnica per la preparazione di omogenati chiarito umano corticale, la separazione delle proteine ​​mediante SDS-PAGE, e antigene-epitopo recupero / 6E10 blotting con anticorpi verso la regione N-terminale del peptide Aβ occidentale. Usando questo protocollo, abbiamo costantemente rilevare Aβ monomeri, dimeri, trimeri, tetrameri, e superiori multimeri peso molecolare nel tessuto corticale da esseri umani con patologia di 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

Abbiamo descritto una tecnica per la preparazione di omogenati chiarito umano corticale, la separazione delle proteine ​​mediante SDS-PAGE, recupero antigene e immunoblotting con un anticorpo per il peptide Aβ. Usando questo protocollo, abbiamo costantemente rilevare Aβ monomerici e multimerica nel tessuto corticale da esseri umani con patologia di Alzheimer.

Rilevazione SDS-PAGE/Immunoblot di multimeri Aβ in Human tessuto corticale omogenati utilizzando Antigen-epitopi recupero

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 Views.  Emory University. Abbiamo descritto una tecnica per la preparazione di omogenati chiarito umano corticale, la separazione delle proteine ​​mediante SDS-PAGE, recupero antigene e immunoblotting con un anticorpo per il peptide Aβ. Usando questo protocollo, abbiamo costantemente rilevare Aβ monomerici e multimerica nel tessuto corticale da esseri umani con patologia di Alzheimer.

Video: 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.

Crioconservazione di tessuto corticale blocchi per la generazione di altamente arricchito Culture neuronale

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

Ricerca

Neuroscienze

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.

Time-lapse immagini dal vivo di relazione clonale cellule progenitrici neurali in via di sviluppo Zebrafish proencefalo

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.

High Sensitivity 5-hydroxymethylcytosine rilevamento in Balb / C tessuto cerebrale

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.

Coerenza tra funzione cerebrale corticale e prestazioni neurocognitive durante condizioni di gravità Cambiato

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.

Generazione di cellule staminali neurali da scartate tessuto fetale umano corticale

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.

Calcio Imaging funzionale nello sviluppo di reti corticali

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.

La quantificazione di filamentosa Actina (F-actina) Puncta nel ratto neuroni corticali

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.

Quantificazione delle endosomi e dei motivi di lisosomi nei neuroni coltivati ​​utilizzando sonde fluorescenti

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.

Visualizzazione dei moduli corticali nelle cortecce dei mammiferi appiattite

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.

Modellazione statistica della connettività corticale mediante elettroencefalogrammi non invasivi

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