Name: the detection of 5-hydroxymethylcytosine in neural stem cells and brains of mice
Uploaded: 2019-09-19T14:00:00
Description: Watch this Scientific Journal Video about The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice at JoVE.com

XL部分得到了中国国家重点研发项目（2017YFE0196600）和国家自然科学基金（授权号31771395，31571518）的支持。Q.S.得到了国家重点研发项目（2017YFC1001703）和浙江省重点研发项目（2017C03009）的支持。W.X.得到了浙江省自然科学基金（LY18H020002）和浙江省科技厅（2017C37057）的支持。

所有动物程序均已通过浙江大学动物伦理委员会批准。
1. 成人神经干细胞和神经元的培养
<ol><li>分离成人神经干细胞从成人（8-10周大）C57/BL6雄性小鼠前脑，如前31，32所述。</li>
	<li>在DMEM/F-12培养基培养的成人神经干细胞中含有20纳克/mL FGF-2、20纳克/mL EGF、2%B27补充剂、1%抗生素抗霉菌和2mM L-谷氨酰胺，在37°C的5%CO2培养箱中。</...

<ol>
	<li>Tan, L., Shi, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tet+family+proteins+and+5-hydroxymethylcytosine+in+development+and+disease.">Tet family proteins and 5-hydroxymethylcytosine in development and disease.</a> Development. 139 (11), 1895-1902 (2012).</li><li>Yao, B., 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=Epigenetic+mechanisms+in+neurogenesis.">Epigenetic mechanisms in neurogenesis.</a> Nature Reviews Neuroscience. 17 (9), 537-549 (2016).</li><li>Day, J. J., Sweatt, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+and+memory+formation.">DNA methylation and memory formation.</a> Nature Neuroscience. 13 (11), 1319-1323 (2010).</li><li>Wu, X. J., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TET-mediated+active+DNA+demethylation:+mechanism,+function+and+beyond.">TET-mediated active DNA demethylation: mechanism, function and beyond.</a> Nature Reviews Genetics. 18 (9), 517-534 (2017).</li><li>Sun, W. J., Guan, M. X., Li, X. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Hydroxymethylcytosine-Mediated+DNA+Demethylation+in+Stem+Cells+and+Development.">5-Hydroxymethylcytosine-Mediated DNA Demethylation in Stem Cells and Development.</a> Stem Cells and Development. 23 (9), 923-930 (2014).</li><li>Li, S., Mason, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pivotal+regulatory+landscape+of+RNA+modifications.">The pivotal regulatory landscape of RNA modifications.</a> Annual Review of Genomics and Human Genetics. 15, 127-150 (2014).</li><li>Hwang, J. Y., Aromolaran, K. A., Zukin, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+field+of+epigenetics+in+neurodegeneration+and+neuroprotection.">The emerging field of epigenetics in neurodegeneration and neuroprotection.</a> Nature Reviews Neuroscience. 18 (6), 347-361 (2017).</li><li>Kriaucionis, S., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nuclear+DNA+base+5-hydroxymethylcytosine+is+present+in+Purkinje+neurons+and+the+brain.">The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain.</a> Science. 324 (5929), 929-930 (2009).</li><li>Tahiliani, M., 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=Conversion+of+5-Methylcytosine+to+5-Hydroxymethylcytosine+in+Mammalian+DNA+by+MLL+Partner+TET1.">Conversion of 5-Methylcytosine to 5-Hydroxymethylcytosine in Mammalian DNA by MLL Partner TET1.</a> Science. 324 (5929), 930-935 (2009).</li><li>Guo, J. U., 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=Neuronal+activity+modifies+the+DNA+methylation+landscape+in+the+adult+brain.">Neuronal activity modifies the DNA methylation landscape in the adult brain.</a> Nature Neuroscience. 14 (10), 1345-1351 (2011).</li><li>Feng, J., 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=Dnmt1+and+Dnmt3a+maintain+DNA+methylation+and+regulate+synaptic+function+in+adult+forebrain+neurons.">Dnmt1 and Dnmt3a maintain DNA methylation and regulate synaptic function in adult forebrain neurons.</a> Nature Neuroscience. 13 (4), 423-430 (2010).</li><li>Jaenisch, R., Bird, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+regulation+of+gene+expression:+how+the+genome+integrates+intrinsic+and+environmental+signals.">Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals.</a> Nature Geneticset. , 245-254 (2003).</li><li>Szulwach, K. E., 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=5-hmC-mediated+epigenetic+dynamics+during+postnatal+neurodevelopment+and+aging.">5-hmC-mediated epigenetic dynamics during postnatal neurodevelopment and aging.</a> Nature Neuroscience. 14 (12), (2011).</li><li>Song, C. X., 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=Selective+chemical+labeling+reveals+the+genome-wide+distribution+of+5-hydroxymethylcytosine.">Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine.</a> Nature Biotechnology. 29 (1), 68-72 (2011).</li><li>Shu, L. Q., 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=Genome-wide+alteration+of+5-hydroxymenthylcytosine+in+a+mouse+model+of+Alzheimer's+disease.">Genome-wide alteration of 5-hydroxymenthylcytosine in a mouse model of Alzheimer's disease.</a> BMC Genomics. 17, (2016).</li><li>Cruvinel, E., 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=Reactivation+of+maternal+SNORD116+cluster+via+SETDB1+knockdown+in+Prader-Willi+syndrome+iPSCs.">Reactivation of maternal SNORD116 cluster via SETDB1 knockdown in Prader-Willi syndrome iPSCs.</a> Human molecular genetics. 23 (17), 4674-4685 (2014).</li><li>Bernstein, A. I., 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=5-Hydroxymethylation-associated+epigenetic+modifiers+of+Alzheimer's+disease+modulate+Tau-induced+neurotoxicity.">5-Hydroxymethylation-associated epigenetic modifiers of Alzheimer's disease modulate Tau-induced neurotoxicity.</a> Human Molecular Genetics. 25 (12), 2437-2450 (2016).</li><li>Wang, F. L., 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=Genome-wide+loss+of+5-hmC+is+a+novel+epigenetic+feature+of+Huntingtons+disease.">Genome-wide loss of 5-hmC is a novel epigenetic feature of Huntingtons disease.</a> Human Molecular Genetics. 22 (18), 3641-3653 (2013).</li><li>Yu, H., 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=Tet3+regulates+synaptic+transmission+and+homeostatic+plasticity+via+DNA+oxidation+and+repair.">Tet3 regulates synaptic transmission and homeostatic plasticity via DNA oxidation and repair.</a> Nature Neuroscience. 18 (6), 836-843 (2015).</li><li>Wu, H., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversing+DNA+methylation:+mechanisms,+genomics,+and+biological+functions.">Reversing DNA methylation: mechanisms, genomics, and biological functions.</a> Cell. 156 (1-2), 45-68 (2014).</li><li>Lister, 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=Global+epigenomic+reconfiguration+during+mammalian+brain+development.">Global epigenomic reconfiguration during mammalian brain development.</a> Science. 341 (6146), 1237905 (2013).</li><li>Inoue, A., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-Dependent+Loss+of+5-Hydroxymethylcytosine+in+Mouse+Preimplantation+Embryos.">Replication-Dependent Loss of 5-Hydroxymethylcytosine in Mouse Preimplantation Embryos.</a> Science. 334 (6053), 194 (2011).</li><li>Pastor, W. 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=Genome-wide+mapping+of+5-hydroxymethylcytosine+in+embryonic+stem+cells.">Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells.</a> Nature. 473 (7347), 394-397 (2011).</li><li>Ito, S., 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=Role+of+Tet+proteins+in+5mC+to+5hmC+conversion,+ES-cell+self-renewal+and+inner+cell+mass+specification.">Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification.</a> Nature. 466 (7310), (2010).</li><li>Wang, T., 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=Genome-wide+DNA+hydroxymethylation+changes+are+associated+with+neurodevelopmental+genes+in+the+developing+human+cerebellum.">Genome-wide DNA hydroxymethylation changes are associated with neurodevelopmental genes in the developing human cerebellum.</a> Human Molecular Genetics. 21 (26), 5500-5510 (2012).</li><li>Song, C. X., 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=Selective+chemical+labeling+reveals+the+genome-wide+distribution+of+5-hydroxymethylcytosine.">Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine.</a> Nature Biotechnology. 29 (1), 68-72 (2011).</li><li>Wang, T., 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=Subtelomeric+hotspots+of+aberrant+5-hydroxymethylcytosine-mediated+epigenetic+modifications+during+reprogramming+to+pluripotency.">Subtelomeric hotspots of aberrant 5-hydroxymethylcytosine-mediated epigenetic modifications during reprogramming to pluripotency.</a> Nature Cell Biology. 15 (6), 700-711 (2013).</li><li>Li, X., 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=Ten-eleven+translocation+2+interacts+with+forkhead+box+O3+and+regulates+adult+neurogenesis.">Ten-eleven translocation 2 interacts with forkhead box O3 and regulates adult neurogenesis.</a> Nature Communications. 8, 15903 (2017).</li><li>Szulwach, K. E., 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=5-hmC-mediated+epigenetic+dynamics+during+postnatal+neurodevelopment+and+aging.">5-hmC-mediated epigenetic dynamics during postnatal neurodevelopment and aging.</a> Nature Neuroscience. 14 (12), 1607-1616 (2011).</li><li>Tao, H., 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=The+Dynamic+DNA+Demethylation+during+Postnatal+Neuronal+Development+and+Neural+Stem+Cell+Differentiation.">The Dynamic DNA Demethylation during Postnatal Neuronal Development and Neural Stem Cell Differentiation.</a> Stem Cells International. 2018, 2186301 (2018).</li><li>Li, X. K., 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=Ten-eleven+translocation+2+interacts+with+forkhead+box+O3+and+regulates+adult+neurogenesis.">Ten-eleven translocation 2 interacts with forkhead box O3 and regulates adult neurogenesis.</a> Nature Communications. 8, (2017).</li><li>Li, X., 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=Epigenetic+regulation+of+the+stem+cell+mitogen+Fgf-2+by+Mbd1+in+adult+neural+stem/progenitor+cells.">Epigenetic regulation of the stem cell mitogen Fgf-2 by Mbd1 in adult neural stem/progenitor cells.</a> Journal of Biological Chemistry. 283 (41), 27644-27652 (2008).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nature Protocols. 1 (5), 2406-2415 (2006).</li></ol>

表观遗传修饰在大脑发育、成熟和功能中起着至关重要的作用。作为DNA修饰的稳定标记，动态5-hmC对行为适应、神经元活动有反应，与基因表达呈正相关;因此，它参与大脑的正常功能和神经紊乱4。为了探索它在细胞和组织中的功能，有必要检测5-hmC的存在，并比较治疗前后的水平。在这里，我们演示了两种检测细胞和组织5-hmC的便捷方法，这些方法可以在实验室中使用普通设备?...

表观遗传修饰，包括DNA修饰，组蛋白修饰和RNA修饰，已被证明在不同的生物过程和疾病1，2，3， 4,5,6,7.长期以来，DNA甲基化（即5-甲基细胞氨酸（5-mC））一直被视为高度稳定的表观遗传标记，无法在基因组中进一步修饰。最近，发现TET（十-十一位）家族蛋白（TET1、TET2和TET38、9）可氧化至5-羟基甲基细胞氨酸（5-hmC）。进一步研究表明，5-hmC可以作为一个稳定的标记，并通过调节基因表达4，10，11，12发挥生物作用。
目前的证据表明，5-hmC在神经元组织/细胞中相对于哺乳动物的其他类型的组织高度丰富，并在神经元发育13、14过程中表现出动态特征。在神经元系统中，5-hmC介导表观遗传修饰在调节神经干细胞、神经元活动、学习和记忆方面起着重要作用，并涉及多种神经系统疾病，包括雷特综合征、自闭症、阿尔茨海默氏症疾病，亨廷顿舞蹈症等2，13，15，16，17，18，19，20。
有几种方法可以检测细胞和组织中的5-hmC14、21、22、23、24。在这里，我们描述了两种方法来检测5-hmC的存在，并量化5-hmC的全球水平：免疫荧光染色和DNA点斑。这两种方法方便而敏感，在以前的研究中已成功地使用了25、26、27、28、29、30。这两种方法的关键步骤是DNA变性。对于 5-hmC 的免疫荧光染色，需要对具有 1 M HCl 的样品进行预处理。对于 5-hmC 点斑点，使用 NaOH 溶液执行 DNA 变性。这两种方法以及下一代测序是研究5-hmC功能的非常有用的工具。

<table>
	<tbody>
		<tr>
			<td>4&#39;-6-diamidino-2-phenylindole (DAPI )</td>
			<td>Sigma-Aldrich</td>
			<td>D8417</td>
		</tr>
		<tr>
			<td>Adobe Photoshop software</td>
			<td>Adobe Inc.</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-rabbit IgG</td>
			<td>Thermo Fisher</td>
			<td>A11008</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 goat anti-mouse IgG</td>
			<td>Thermo Fisher</td>
			<td>A11001</td>
		</tr>
		<tr>
			<td>anti-5-hydroxymethylcytosine</td>
			<td>Active Motif</td>
			<td>39769</td>
		</tr>
		<tr>
			<td>anti-NeuN</td>
			<td>Millipore</td>
			<td>MAB377</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>12587-010</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>12580-010</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
		</tr>
		<tr>
			<td>Cryostat microtome</td>
			<td>Leica</td>
			<td>CM1950</td>
		</tr>
		<tr>
			<td>DMEM/F-12 medium</td>
			<td>OmegaScientific</td>
			<td>DM25</td>
		</tr>
		<tr>
			<td>epidermal growth factor</td>
			<td>PeproTech</td>
			<td>100-15</td>
		</tr>
		<tr>
			<td>Fibroblast growth factor-basic</td>
			<td>PeproTech</td>
			<td>100-18B</td>
		</tr>
		<tr>
			<td>forskolin</td>
			<td>Sigma-Aldrich</td>
			<td>F6886</td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Thermo</td>
			<td>35050061</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-149</td>
		</tr>
		<tr>
			<td>neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
		</tr>
		<tr>
			<td>normal goat serum</td>
			<td>Vector Laboratories</td>
			<td>Z0325</td>
		</tr>
		<tr>
			<td>nylon membrane (Hybond&#8482;-N+ )</td>
			<td>Amersham Biosciences</td>
			<td>RPN303B</td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>Leica</td>
			<td>14020108926</td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td>phenol: chloroform: isoamyl alcohol (25: 24:1 )</td>
			<td>Sigma-Aldrich</td>
			<td>516726</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma</td>
			<td>P0899-10</td>
		</tr>
		<tr>
			<td>proteinase K</td>
			<td>VVR</td>
			<td>39450-01-6</td>
		</tr>
		<tr>
			<td>retinoic acid</td>
			<td>Sigma-Aldrich</td>
			<td>R2625</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Solarbio</td>
			<td>T8210</td>
		</tr>
	</tbody>
</table>

the detection of 5-hydroxymethylcytosine in neural stem cells and brains of mice

在哺乳动物基因组中已经发现了多个DNA修饰。其中，对5-甲基细胞氨酸和5-羟基甲基细胞氨酸介导表观遗传机制进行了深入的研究。5-羟基甲基细胞氨酸在大脑胚胎发育和产后发育过程中表现出动态特征，在基因表达中起着调节作用，并涉及多种神经系统疾病。在这里，我们描述了详细的方法，包括免疫荧光染色和DNA点缀，以检测培养细胞和小鼠脑组织中5-羟基甲基细胞氨酸。

为了揭示成年小鼠海马区5-hmC的分布，我们用抗体对神经元细胞（NeuN）和5-hmC进行了免疫荧光。在海马区，5-hmC与神经元细胞标记NeuN（图1A-H）共同本地化良好，表明神经元中5-hmC的富集。
为了确定神经元发育过程中的5-hmC动力学，首先使用从增殖和分化的成人神经干细胞（NSCs）中分离出的DNA样本进行点斑点。点斑点结果显示，在NSC分化期间?...

Watch this Scientific Journal Video about The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice at JoVE.com

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

在这里，我们提出了一个协议，利用免疫荧光染色和DNA点印法检测细胞和脑组织中5-羟基甲基细胞氨酸。

小鼠神经干细胞和脑中5-羟基甲基细胞氨酸的检测

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

在这里，我们提出了一个方案，利用免疫荧光、新方法和DNA点斑点检测细胞和脑组织中5-羟基霉素。5-羟基霉素，即5hmC，减轻表观遗传修饰，在调节基因表达和参与多种神经系统疾病方面起着重要作用。实际上，我们的方法可用于检测多个细胞和组织中的5hmC。它很方便，可以在实验室中使用通用设备执行。首先将鼠标面朝上放在塑料板上，然后用胶带固定每个肢体。使用手术剪刀切开皮肤和肌肉，并打开胸腔。用精细的手术剪刀暴露心脏并切断右心房的一小部分。使用 10 毫升一次性消毒注射器，将鼠标从左心室中用冷 PBS 排出。然后，用 4%PFA 将鼠标注入，直到鼠标变硬。然后，用骨钳打开头骨，取出大脑。将大脑放入一个15毫升的离心管中，里面装满了5毫升4%PFA的离心机，并将其储存在4摄氏度。24小时后，将大脑转移到30%蔗糖溶液中，并留在4摄氏度下进行完全脱水。在切块之前，将大脑用最佳的切割温度复合物吸收，并冷却到零下20摄氏度至少一小时。使用低温计子微原子进行厚度为20至40微米的分段大脑样本。将部分收集到 PBS 中，并在摄氏四度时存储。拿起感兴趣的大脑部分，并把它们放入一个24井板与PBS。在摇床上用 PBS 清洗部分 10 分钟。取出PBS，在37摄氏度下用预热的一摩尔盐酸处理30分钟。盐酸具有腐蚀性，请准备在化学保持中。然后，用PBS清洗样品三次五分钟，用含有3%正常山羊血清和0.1%Triton X-100的PBS进行阻断。将样品放在室温下摇床上一小时。向各部分添加特定的原抗体，并在四摄氏度的摇床上孵育过夜。第二天，将样品拿出来，在室温下留一小时。用PBS清洗样品三次，并添加二次抗体。用铝盖住盘子，在摇动时在室温下保持一小时。孵育后，用PBS清洗样品三次，然后将它们安装到幻灯片上。加入 100 到 150 微升的防褪色安装介质，用优质盖玻璃盖住，用指甲油密封。用普通显微镜或共体显微镜成像大脑部分。要分离基因组DNA，首先解剖冰冷盘上的海马、皮层和小脑组织。加入一毫升的DNA解液缓冲液，研磨组织。将样品转移到干净的微离心管中，每600微升解液缓冲液加入250微克蛋白质酶-K。然后，每样本加入约50微克的RNase A，在37摄氏度下孵育至少12小时。孵育后，加入同等体积的苯酚氯仿异丙酰醇，并彻底混合。根据手稿指示将混合物离心，然后将上一代转移到新管中。在上清液中加入 600 微升氯仿，根据手稿指示彻底混合和离心机。将上流液转移到新管中，并添加 500 微升异丙醇。彻底混合并再次离心，沉淀DNA。离心后，丢弃上经剂，然后根据手稿指示用70%乙醇清洗DNA颗粒。空气完全干燥DNA颗粒，然后溶解在Tris-HCl缓冲液中。在开始之前，根据手稿指示准备所需的溶液和样品混合物。将DNA样本加热至100摄氏度10分钟，然后放在冰上。切下尼龙膜的适当尺寸，然后用 6X SSC 冲洗。将膜放在点印装置上，然后连接真空泵。在膜上发现六微升点，在 80 摄氏度下混合 30 分钟。然后，用无脂牛奶和Tris缓冲盐水（TBS）阻断样品膜一小时。将多克隆兔抗5-羟基霉素抗体加入膜，并在4摄氏度下孵育过夜。第二天，将样品膜留在室温下一小时，然后用TBS洗涤膜三次。用抗兔二级抗体孵育膜30分钟，然后用TBS洗涤三次。该协议可用于在成年小鼠的海马体中可视化5-羟基霉素，或5hmC。5hmC 的免疫荧光与神经元细胞的抗体，揭示了神经元中 5hmC 的富集。在神经元发育过程中，5hmC的动力学可以通过从增殖和分化的成人神经干细胞中分离出的DNA样本进行点-斑点测定来测定。在分化过程中，5hmC的全球水平显著增加，神经元中5hmC的水平高于神经干细胞。这种方法的关键步骤是确保DNA样本完全变性。

Research

JoVE Journal

Neuroscience

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

成年鼠神经干细胞的分离和扩大使用神经干含量

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

科研

神经科学

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

BALB / C脑组织中的高灵敏度检测5 - hydroxymethylcytosine

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

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

小鼠神经干细胞移植手术与器官的诱向性鼠肝炎病毒感染小鼠的脊髓

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

检测内源NADH的荧光双光子成像的小鼠大脑皮层Microregional缺氧

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

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

从小鼠胚胎神经管的神经嵴细胞的分离和培养

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

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

一个鼠标，两种文化：隔离和成人神经的培养干细胞从单个小鼠的两个神经源性区

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

从人多能神经嵴前体细胞的无饲养推导干细胞

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

建模星形细胞瘤发病机制<em&gt;在体外</em&gt;和<em&gt;体内</em&gt;使用星形胶质细胞或条件，遗传工程小鼠神经干细胞

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Enumeration of Neural Stem Cells Using Clonal Assays

使用克隆测定神经干细胞的计数。

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

分离和成人神经干细胞的培养从鼠标Subcallosal区

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...