Name: quantitation of &gamma;h2ax foci in tissue samples
Uploaded: 2010-06-28T17:33:00
Description: Watch this Scientific Journal Video about Quantitation of ÃŽÂ³H2AX Foci in Tissue Samples at JoVE.com

澳大利亚核科学与工程学院的支持，是公认的事实。 TCK的是AINSE奖项的收件人。表观医学实验室是支持由国家卫生和澳大利亚的医学研究理事会（566559）。 LM是由墨尔本研究所（墨尔本大学）和生物医学成像的华润补充奖学金支持。莫纳什显微成像（DRS斯蒂芬科迪ISKA卡迈克尔）的支持是这项工作的宝贵。蒙脱土是奥尔加Sedelnikova博士从美国国立卫生研究院的专家援助和意见表示感谢。

组织<ol><li>肺组织，在模具中嵌入最佳切削温度（OCT）的化合物4583，切片（5μm）的使用CM徕卡1950年的低温恒温器，安装到聚- L -赖氨酸的幻灯片。 </li></ol>冷冻样品在默多克儿童研究所获得安博士劳斯莱斯。肺组织是未经处理的BALB / c小鼠的慢性呼吸道过敏性疾病，显示许多人类哮喘6的病理特征的小鼠模型。默多克儿童研究院坚持实践澳大利亚的关心和用于科研目的的?...

<ol>
	<li>Rogakou, E. P., Boon, C., Redon, C., Bonner, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Megabase+chromatin+domains+involved+in+DNA+double-strand+breaks+in+vivo.">Megabase chromatin domains involved in DNA double-strand breaks in vivo.</a> J. Cell Biol. 146, 905-916 (1999).</li><li>Bonner, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamma+H2AX+and+cancer.">Gamma H2AX and cancer.</a> Nature Reviews Cancer. 8 (12), 957-967 (2008).</li><li>Dickey, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H2AX:+functional+roles+and+potential+applications.">H2AX: functional roles and potential applications.</a> Chromosoma. 118 (6), 683-692 (2009).</li><li>Bhogal, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late+residual+gamma-H2AX+foci+in+murine+skin+are+dose+responsive+and+predict+radiosensitivity+in+vivo.">Late residual gamma-H2AX foci in murine skin are dose responsive and predict radiosensitivity in vivo.</a> Radiat Res. 173 (1), 1-9 (2010).</li><li>Redon, C. E., Dickey, J. S., Bonner, W. M., Sedelnikova, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gamma-H2AX+as+a+biomarker+of+DNA+damage+induced+by+ionizing+radiation+in+human+peripheral+blood+lymphocytes+and+artificial+skin.">gamma-H2AX as a biomarker of DNA damage induced by ionizing radiation in human peripheral blood lymphocytes and artificial skin.</a> Adv Space Res. 43 (8), 1171-1178 (2009).</li><li>Locke, N. R., Royce, S. G., Wainewright, J. S., Samuel, C. S., Tang, M. L., L, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+airway+remodeling+in+acute,+subacute,+and+chronic+models+of+allergic+airways+disease.">Comparison of airway remodeling in acute, subacute, and chronic models of allergic airways disease.</a> Am J Respir Cell Mol Biol. 36 (5), 625-632 (2007).</li><li>Bolte, S. &. a. m. p. ;. a. m. p., Cordelieres, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guided+tour+into+subcellular+colocalization+analysis+in+light+microscopy.">A guided tour into subcellular colocalization analysis in light microscopy.</a> J Microsc. 224 (Pt 3), 213-232 (2006).</li></ol>

这个演示中，我们使用未经处理的BALB / c小鼠，小鼠肺组织的慢性呼吸道过敏性疾病的小鼠模型的目的。我们定量细胞受损的肺相比，未经处理的部分的平均水平略高，每灶数量的差异。具体来说，定量表示6.5元/单元和9.4灶元/单元（每节20细胞人工计数）灶，在未经处理的组织和慢性呼吸道过敏性疾病组织的分别。然而，例如萘，这是众所周知的诱导小鼠肺癌模型的DSBs的DSB诱导剂的使用，会产生...

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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>Bovine Serum Albumin (BSA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A7906</td>
<td>BSA (1%) is used to block any non-specific antibody binding. Primary and secondary antibodies are diluted in BSA.</td>
</tr>
<tr>
<td>PBS (without Ca2+ and Mg2+)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17-517Q</td>
<td>&nbsp;</td>
<tr>
<td>Optimal Cutting Temperature (OCT) Compound 4583</td>
<td>&nbsp;</td>
<td>Tissue-Tek</td>
<td>4583</td>
<td>&nbsp;</td>
<tr>
<td>Superfrost Plus</td>
<td>Polysine slides</td>
<td>Menzel-Gl&auml;ser, Germany</td>
<td>SF41296SP</td>
<td>Electrostatically attracts frozen tissue sections and reduces the need for additional coatings and adhesives.</td>
</tr>
<tr>
<td>Tween 20</td>
<td>Reagent</td>
<td>Calbiochem</td>
<td>655205</td>
<td>Tween 20 (0.5%), is a detergent to permeabilise cells</td>
</tr>
<tr>
<td>Triton X-100</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>T8787</td>
<td>Triton X-100 (0.1%) used to permeabilise cells.</td>
</tr>
<tr>
<td>Paraformaldehyde</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>158127</td>
<td>&nbsp;</td>
<tr>
<td>Anti-phospho-&gamma;H2AX (rabbit polyclonal antibody)</td>
<td>Primary Antibody</td>
<td>Upstate, USA</td>
<td>07-164</td>
<td>Dilution of primary antibody (1:500), in 1% BSA.</td>
</tr>
<tr>
<td>Alexa Fluor 488 goat anti-rabbit IgG (H+L)</td>
<td>Secondary Antibody</td>
<td>Invitrogen</td>
<td>A-11008</td>
<td>Dilution of secondary antibody (1:500), in 1% BSA.</td>
</tr>
<tr>
<td>Ethanol (70%)</td>
<td>&nbsp;</td>
<td>Thermo Fisher</td>
<td>BSPEL316</td>
<td>&nbsp;</td>
<tr>
<td>RNAse A</td>
<td>Reagent</td>
<td>Qiagen </td>
<td>19101</td>
<td>&nbsp;</td>
<tr>
<td>Vectashield PI</td>
<td>Reagent</td>
<td>Vector Laboratories</td>
<td>H-1300</td>
<td>A glycerol based mounting medium containing propidium iodide (a red nuclear stain).This medium must be used with an oil based lense that matches its refractive index. 
Note: DNA stain commonly used: 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). Can only be used with microscopes with the appropriate excitation laser.</td>
</tr>
<tr>
<td>Mini PAP Pen</td>
<td>&nbsp;</td>
<td>Invitrogen, USA</td>
<td>008877</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips (22x50mm)</td>
<td>&nbsp;</td>
<td>Menzel-Gl&auml;ser, Germany</td>
<td>CS2250100</td>
<td>&nbsp;</td>
<tr>
<td>Coplin Jar, glass</td>
<td>&nbsp;</td>
<td>Grale Scientific P/L</td>
<td>1771-OG</td>
<td>&nbsp;</td>
<tr>
<td>Staining Trough</td>
<td>&nbsp;</td>
<td>Grale Scientific P/L</td>
<td>V1991.99</td>
<td>&nbsp;</td>
<tr>
<td>Zeiss LSM 510 Meta Confocal</td>
<td>&nbsp;</td>
<td>Confocal Microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CM Leica 1950 Cryostat</td>
<td>&nbsp;</td>
<td>Leica Microsystems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Metamorph</td>
<td>Software for Imaging analysis</td>
<td>Molecular Devices, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

quantitation of &gamma;h2ax foci in tissue samples

DNA双链断裂（DSB的），特别是致死和遗传毒性病变，可以产生内源性（生理或病理）的进程或外在因素，特别是电离辐射和radiomimetic化合物。磷酸化的组蛋白H2A的变种，H2AX的丝氨酸139残留，高度保守的C -末端SQEY图案，形成γH2AX，是一种早期反应的DNA双链断裂<sup&gt; 1</sup&gt;。这inosito的磷脂，肌醇3 - 激酶（PI3K）的蛋白质家族，共济失调毛细血管扩张症（ATM）的突变，DNA -蛋白激酶催化亚基和ATM和RAD3相关介导的磷酸化事件（ATR）<sup&gt; 2</sup&gt;。整体而言，在形成离散核γH2AX灶DSB诱导的结果，可以很容易地检测和免疫荧光显微镜定量分析<sup&gt; 2</sup&gt;。由于这个标记独特的特异性和灵敏度，分析γH2AX灶导致广泛的生物医学研究中的应用程序，尤其是在放射生物学和核医学，。 γH2AX灶的定量已在电离辐射诱导的DNA双链断裂的情况下在细胞培养系统最广泛的调查。除了从细胞放射敏感性，免疫检测也被用来评估辐射修改化合物的疗效。此外，γH2AX作为分子标记已用于研究各种DSB诱导化合物的疗效，是最近正预示着衰老和疾病的重要标志，尤其是癌症<sup&gt; 3</sup&gt;。此外，免疫为基础的方法已经适应了西装检测和定量γH2AX灶<em&gt;体外</em&gt;<em&gt;在体内</em<sup&gt; 4,5</sup&gt;。在这里，我们展示了一个典型的免疫荧光法在小鼠组织中γH2AX灶的检测和定量。

Watch this Scientific Journal Video about Quantitation of ÃŽÂ³H2AX Foci in Tissue Samples at JoVE.com

Quantitation of ÃƒÅ½Ã‚Â³H2AX Foci in Tissue Samples

定量DNA双链上的γH2AX灶的基础上休息，已经成为一个宝贵的工具，尤其是在放射生物学，组织的放射敏感性和辐射修改化合物的影响的评价。在这里，我们表现出的组织样本中γH2AX灶的定量免疫荧光法的使用。

定量组织样本中的γH2AX灶

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Research

JoVE Journal

Biology

Quantitation of &gamma;H2AX Foci in Tissue Samples

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

Quantification of ÃƒÅ½Ã‚Â³H2AX Foci in Response to Ionising Radiation

电离辐射的反应定量的γH2AX灶

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

科研

生物学

NanoDrop Microvolume Quantitation of Nucleic Acids

NanoDrop微量核酸的定量

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

测量去泛素酶的活性细胞系和组织样品的方法

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

Quantitation of Endothelial Cell Adhesiveness In Vitro

内皮细胞粘附性定量<em&gt;在体外</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

蛋白表达和共定位使用复用免疫组织化学染色和多光谱成像定量

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

混合式切割：用于顽拗植物组织样品的改进切法

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules

将细胞质Foci分类为哺乳动物应激颗粒的方法

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to ...

Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA

人线粒体 DNA 中核苷酸的同时定位与定量

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using ...

Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples

细胞系定量免疫印迹作为常规组织样本中生物标志物蛋白定量免疫荧光定量的标准

Quantification of proteins of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples is important in clinical and research applications. An ...