Name: evaluation of biomarkers in glioma by immunohistochemistry on paraffin-embedded 3d glioma neurosphere cultures
Uploaded: 2019-01-09T15:00:00
Description: Watch this Scientific Journal Video about Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures at JoVE.com

这项工作得到了国家健康研究所/国家神经疾病研究所的支持 & 中风 (nih/nnds) 赠款 r37-ns094804, r01 ns074387, r21-ns091555 至 m. g. c.;nhhninsd 赠款 r01-ns07691、r01-ns082311 和 r01 ns096756 至中华人民共和国;nhhxninsr01-eb022563;1uo1ca224160;神经外科;莉娅的快乐之心 m. g. c. 和 p. l.;和 rna 生物医学赠款 f046166 m. g. c. f. m. 由 f31 nhhxinds-f31 ns103500 支持。j. p. 得到了阿根廷富力体育和教育部研究金的支持。

这里描述的所有方法都已得到密歇根大学动物护理和使用机构委员会 (iacuc) 的批准。
1. 从小鼠模型衍生出的脑肿瘤神经球的产生
<ol><li>在解剖过程之前, 准备神经干细胞培养基 (nsc 培养基: dmem/f12 与 1x b27 补充剂, 1x n2 补充, 1x 诺康和12倍药/抗真菌的 yic-yacce 补充人类重组 egf 和基本 fgf 在浓度为 20 ngml 每个)。在 1.5 ml 管中填充300μl 的 nsc 介质, 并为以后的工作做好准备。</li>
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<ol>
	<li>Louis, D. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+2016+World+Health+Organization+Classification+of+Tumors+of+the+Central+Nervous+System:+a+summary.">The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary.</a> Acta Neuropatholica. 131 (6), 803-820 (2016).</li><li>Hochberg, F. 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=Glioma+diagnostics+and+biomarkers:+an+ongoing+challenge+in+the+field+of+medicine+and+science.">Glioma diagnostics and biomarkers: an ongoing challenge in the field of medicine and science.</a> Expert Review of Molecular Diagnosis. 14 (4), 439-452 (2014).</li><li>Ziegler, A., Koch, A., Krockenberger, K., Grosshennig, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Personalized+medicine+using+DNA+biomarkers:+a+review.">Personalized medicine using DNA biomarkers: a review.</a> Human Genetics. 131 (10), 1627-1638 (2012).</li><li>Ceccarelli, 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=Molecular+Profiling+Reveals+Biologically+Discrete+Subsets+and+Pathways+of+Progression+in+Diffuse+Glioma.">Molecular Profiling Reveals Biologically Discrete Subsets and Pathways of Progression in Diffuse Glioma.</a> Cell. 164 (3), 550-563 (2016).</li><li>Cancer Genome Atlas Research, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive,+Integrative+Genomic+Analysis+of+Diffuse+Lower-Grade+Gliomas.">Comprehensive, Integrative Genomic Analysis of Diffuse Lower-Grade Gliomas.</a> The New England Journal of Medicine. 372 (26), 2481-2498 (2015).</li><li>Mackay, 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=Integrated+Molecular+Meta-Analysis+of+1,000+Pediatric+High-Grade+and+Diffuse+Intrinsic+Pontine+Glioma.">Integrated Molecular Meta-Analysis of 1,000 Pediatric High-Grade and Diffuse Intrinsic Pontine Glioma.</a> Cancer Cell. 32 (4), 520-537 (2017).</li><li>Koschmann, C., 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=ATRX+loss+promotes+tumor+growth+and+impairs+nonhomologous+end+joining+DNA+repair+in+glioma.">ATRX loss promotes tumor growth and impairs nonhomologous end joining DNA repair in glioma.</a> Science Translational Medicine. 8 (328), (2016).</li><li>Ward, T. 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=Biomarkers+of+apoptosis.">Biomarkers of apoptosis.</a> British Journal of Cancer. 99 (6), 841-846 (2008).</li><li>Gil, J., Pesz, K. A., Sasiadek, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=May+autophagy+be+a+novel+biomarker+and+antitumor+target+in+colorectal+cancer.">May autophagy be a novel biomarker and antitumor target in colorectal cancer.</a> Biomarkers in Medicine. 10 (10), 1081-1094 (2016).</li><li>Williams, G. H., Stoeber, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+cycle+and+cancer.">The cell cycle and cancer.</a> Journal of Pathology. 226 (2), 352-364 (2012).</li><li>Preusser, 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=Ki67+index+in+intracranial+ependymoma:+a+promising+histopathological+candidate+biomarker.">Ki67 index in intracranial ependymoma: a promising histopathological candidate biomarker.</a> Histopathology. 53 (1), 39-47 (2008).</li><li>Trepant, A. 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=Identification+of+OLIG2+as+the+most+specific+glioblastoma+stem+cell+marker+starting+from+comparative+analysis+of+data+from+similar+DNA+chip+microarray+platforms.">Identification of OLIG2 as the most specific glioblastoma stem cell marker starting from comparative analysis of data from similar DNA chip microarray platforms.</a> Tumour Biology. 36 (3), 1943-1953 (2015).</li><li>Calinescu, A. 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=Transposon+mediated+integration+of+plasmid+DNA+into+the+subventricular+zone+of+neonatal+mice+to+generate+novel+models+of+glioblastoma.">Transposon mediated integration of plasmid DNA into the subventricular zone of neonatal mice to generate novel models of glioblastoma.</a> Journal of Visualized Experiments. (96), (2015).</li><li>Wiesner, S. 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=De+novo+induction+of+genetically+engineered+brain+tumors+in+mice+using+plasmid+DNA.">De novo induction of genetically engineered brain tumors in mice using plasmid DNA.</a> Cancer Research. 69 (2), 431-439 (2009).</li><li>Xie, Y., 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+Human+Glioblastoma+Cell+Culture+Resource:+Validated+Cell+Models+Representing+All+Molecular+Subtypes.">The Human Glioblastoma Cell Culture Resource: Validated Cell Models Representing All Molecular Subtypes.</a> EBioMedicine. 2 (10), 1351-1363 (2015).</li><li>Vescovi, A. L., Galli, R., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+tumour+stem+cells.">Brain tumour stem cells.</a> Nature Reviews Cancer. 6, 425 (2006).</li><li>Dirks, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+tumor+stem+cells:+bringing+order+to+the+chaos+of+brain+cancer.">Brain tumor stem cells: bringing order to the chaos of brain cancer.</a> Journal of Clinical Oncology. 26 (17), 2916-2924 (2008).</li><li>Lenting, K., Verhaak, R., Ter Laan, M., Wesseling, P., Leenders, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioma:+experimental+models+and+reality.">Glioma: experimental models and reality.</a> Acta Neuropathologica. 133 (2), 263-282 (2017).</li><li>Koh, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+cells+for+microscopy+using+cytospin.">Preparation of cells for microscopy using cytospin.</a> Methods in Enzymology. 533, 235-240 (2013).</li><li>Hasselbach, L. 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=Optimization+of+high+grade+glioma+cell+culture+from+surgical+specimens+for+use+in+clinically+relevant+animal+models+and+3D+immunochemistry.">Optimization of high grade glioma cell culture from surgical specimens for use in clinically relevant animal models and 3D immunochemistry.</a> Journal of Visualized Experiments. (83), e51088 (2014).</li><li>Pileri, S. 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=Antigen+retrieval+techniques+in+immunohistochemistry:+comparison+of+different+methods.">Antigen retrieval techniques in immunohistochemistry: comparison of different methods.</a> The Journal of Pathology. 183 (1), 116-123 (1997).</li></ol>

本文详细介绍了一种对石蜡嵌入胶质瘤 ns 进行免疫组织化学的通用和可重复的方法, 该方法保持了原位生长在大脑中的肿瘤细胞的特征三维结构。与使用镀金或塑料表面的细胞相比, 该技术具有以下优点: i) 保存 ns 的三维结构;ii) 在分析蛋白质表达之前, 避免细胞离解的应激;(iii) 从基因工程肿瘤 ns 中淬火任何内源性荧光体表达, 从而在荧光光谱中进行染色;和 iv) 长期储存。
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胶质瘤是根据世界卫生组织1的表型和基因型特征分类的中枢神经系统的主要实体瘤。这种分类包括司机突变的存在或不存在, 这些突变代表了一个有吸引力的生物标志物来源 2。生物标志物是生物特性, 可以测量和评估, 以表明正常和病理过程, 以及药理反应的治疗干预3。生物标志物可以在胶质瘤的肿瘤组织和细胞中检测到, 使其能够在不同的方面进行生物表征。胶质瘤生物标志物的一些例子包括突变的异氰酸酯脱氢酶 1 (idh1), 这是一种与预后较好相关的低度胶质瘤的突变酶特征4。突变 idh1 常与 tp53 和α-地中海贫血综合征 x-连锁 (atrx) 失活突变结合表达, 定义了特定的胶质瘤亚型 4,5。atrx 失活突变也发生在44% 的儿童高级胶质瘤 2,6, 并已与攻击性肿瘤和基因组不稳定6,7。此外, 在组织内 h3 基因 h3f3a 或 hist1hb 基因 1, 6 中, 根据 k27m 突变的存在或不存在, 在亚组中对小儿弥漫性内脑桥胶质瘤 (dipg) 进行分化。因此, 分子特征的引入允许将胶质瘤作为单独的实体进行细分、研究和治疗, 这将更允许为每个亚型开发更有针对性的疗法。此外, 生物标志物的分析可用于评估不同的生物过程, 如凋亡8、自体吞噬9、细胞周期进展 10、细胞增殖11和细胞分化12.
基因工程动物模型, 窝藏人类癌症中存在的遗传病变是至关重要的信号路径的研究, 调解疾病的进展。我们的实验室已经实施了使用睡眠美容 (sb) 转相酶系统, 以开发基因工程小鼠模型的胶质瘤窝藏特定的突变, 重述人类胶质瘤亚型13,14。这些基因工程小鼠模型用于生成肿瘤衍生 ns, 使其能够在三维系统中进行体外研究, 反映了原位15肿瘤中存在的显著特征。此外, 将 ns 移植到小鼠体内可以产生继发性肿瘤, 这些肿瘤保留原发肿瘤的特征, 并模仿相应原发肿瘤15的组织病理学、基因组和表型特性。
在无血清培养中, 具有干细胞特性的脑肿瘤细胞可以得到丰富, 在表皮生长因子 (egf) 和成纤维细胞生长因子 (fgf) 的存在下, 它们可以作为单一细胞衍生的菌落 (如 ns 培养) 生长。在这种选择性培养系统中, 大多数分化或分化的细胞会迅速死亡, 而干细胞则分裂并形成细胞簇。这使得 ns 培养保持胶质瘤肿瘤的特点16,17,18。胶质瘤 ns 可用于评估肿瘤生物学的几个方面, 包括分析生物标志物, 在诊断, 预后, 分类, 肿瘤进展状态, 细胞分化状态, 细胞分化状态。在这里, 我们详细介绍了生成胶质瘤 ns 的协议, 并将3d 培养物嵌入石蜡中, 用于 ihc 染色。固定和嵌入胶质瘤 ns 的一个优点是, 与传统的细胞旋旋法相比, ns 的形态保持得更好,其中胶质瘤 ns 受到细胞离解的压力操纵, 并变得扁平。此外, 嵌入抑制任何内源性荧光体表达从基因工程肿瘤 ns, 使染色整个荧光光谱。该方法的总体目标是通过石蜡包埋过程保存胶质瘤细胞的三维结构, 并利用免疫组织化学对胶质瘤神经球进行表征。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Coated microtome blade HP35</td>
			<td style="width:191px;">Thermo Scientific</td>
			<td style="width:221px;">3150734</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microtome RM 2135</td>
			<td style="width:191px;">Leica</td>
			<td style="width:221px;">MR2135</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Formalin solution, neutral buffered, 10%</td>
			<td style="width:191px;">Sigma-aldrich</td>
			<td>HT501128-4L</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:305px;">Rabbit polyclonal anti-ATRX,</td>
			<td style="width:191px;">Santa Cruz Biotechnology</td>
			<td style="width:221px;">sc-15408</td>
			<td>IHC, 1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Rabbit polyclonal anti-Ki-67</td>
			<td style="width:191px;">Abcam</td>
			<td style="width:221px;">Ab15580</td>
			<td>IHC, 1:1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rabbit polyclonal anti-OLIG2</td>
			<td>Millipore</td>
			<td>AB9610</td>
			<td>IHC, 1:500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat polyclonal anti-rabbit biotin-conjugated</td>
			<td style="width:191px;">Dako</td>
			<td style="width:221px;">E0432</td>
			<td>IHC, 1:1000 dilution</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:305px;">Vectastain Elite ABC HRP kit</td>
			<td style="width:191px;">Vector Laboratories Inc</td>
			<td style="width:221px;">PK-6100</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">BETAZOID DAB CHROMOGEN KIT</td>
			<td style="width:191px;">Biocare medical</td>
			<td style="width:221px;">BDB2004 L/price till 12/18</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">N-2 Supplement</td>
			<td style="width:191px;">ThermoFisher</td>
			<td style="width:221px;">17502048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">N-27 Supplement</td>
			<td style="width:191px;">ThermoFisher</td>
			<td style="width:221px;">A3582801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Accutase&#174; Cell Detachment Solution</td>
			<td style="width:191px;">Biolegend</td>
			<td style="width:221px;">423201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">AGAROSE LE</td>
			<td style="width:191px;">GoldBio</td>
			<td style="width:221px;">A-201-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Genesee Sc. Corporation</td>
			<td style="width:191px;">Olympus 15 ml</td>
			<td style="width:221px;">21-103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Genesee Sc. Corporation</td>
			<td style="width:191px;">TC-75 treated Flask</td>
			<td style="width:221px;">25-209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Genesee Sc. Corporation</td>
			<td style="width:191px;">TC-25 treated Flask</td>
			<td style="width:221px;">25-207</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">DMEM/F12</td>
			<td style="width:191px;">Gibco</td>
			<td style="width:221px;">11330-057</td>
			<td>NS media</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:305px;">HBSS</td>
			<td style="width:191px;">GibcoTM</td>
			<td style="width:221px;">14175-103</td>
			<td>balanced salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">C57BL/6</td>
			<td style="width:191px;">Taconic</td>
			<td style="width:221px;">B6-f</td>
			<td>C57BL/6 mouse</td>
		</tr>
	</tbody>
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evaluation of biomarkers in glioma by immunohistochemistry on paraffin-embedded 3d glioma neurosphere cultures

胶质瘤蛋白表达分析与胶质瘤病理学研究有关。许多蛋白质被描述为生物标志物, 在诊断、预后、分类、肿瘤进展状态和细胞分化状态等方面都有应用。这些生物标志物的分析也有助于表征胶质瘤患者和胶质瘤模型产生的肿瘤神经球 (ns)。肿瘤 ns 提供了一个有价值的体外模型来评估不同的特征, 从中获得, 并能更准确地反映胶质瘤生物学。在这里, 我们描述了一个详细的方法来分析生物标志物在肿瘤 ns 使用免疫组织化学 (ihc) 石蜡嵌入肿瘤 ns。

为了监测肿瘤的发展和评估肿瘤是否达到生成 ns 所需的大小, 我们使用生物发光分析了肿瘤发出的发光。这使得通过荧光素酶的发光信号研究幼崽的转导效率和肿瘤进展 (图 1a和1b)。当肿瘤大小达到 1 x 10 7 光子/ponss\ cm/sr (图 1b) 时, 大脑可以被处理为 ns 生成.该系统的另一个优点是, ...

Watch this Scientific Journal Video about Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures at JoVE.com

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

作为3d 培养体生长的神经球是研究胶质瘤生物学的有力工具。在这里, 我们提出了一个执行免疫组织化学的协议, 同时保持三维结构的胶质瘤神经球通过石蜡嵌入。这种方法可以描述胶质瘤神经球的性质, 如干细胞和神经分化。

石蜡包埋三维胶质瘤神经球培养的免疫组织化学评价胶质瘤生物标志物

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

该协议是重要的，因为可用于研究胶质瘤生物标志物的存在的方法，保持肿瘤神经球的3D结构。该协议的主要优点是，神经球的形态与细胞平方法相比，保持细胞平的方法，其中细胞受到压力操纵，并变得扁平。这种方法可以应用于任何其他组织培养系统，其中细胞在3D培养和结构的维护是重要的。首先，培养肿瘤神经球或NS在T-25烧瓶，直到细胞汇合。在 15 毫升锥形管中选择肿瘤 NS。在 300 Gs 下使用 NS 进行 5 分钟。在此之后，将颗粒重新悬浮在10%甲醛的一毫升中，并在室温下孵育管子10分钟。在重新悬挂的 NS 中加入五毫升的 HBSS。如前所述，将细胞进行浸渍。将含有颗粒的管子放在冰上。将洗涤的 NS 颗粒重新悬浮在 500 微升中，其中含有 2% 的热液体 agarose。并混合通过温和的移液或搅拌。将阿加罗斯嵌入的 NS 放在冰上，直到加糖聚合。然后取出一块加糖，拿着 NS。将一块阿加罗斯放入盒式磁带中进行组织处理。将水浴设置为 42 摄氏度。并小心地将刀片插入显微原子中。然后将石蜡块放入显微原子中。用一只手向下按控制机器左侧每个部分厚度的操纵杆。将操纵杆按至第二个停止，将微切片设置为 30 微米厚度。转动粗手轮开始修剪块。样品表面几乎暴露后，将控制厚度的操纵杆设置为 5 或 10 微米。到达样品表面时停止修剪。将冰盒装满冰块，并有足够的水保护石蜡块，防止其直接接触冰。丢弃旧的微原子刀片，并插入一个新的进行剖切。用纱布擦干石蜡块，放在微原子上。转动粗手轮开始切截石蜡。然后使用潮湿的画笔将石蜡带转移到 42 摄氏度的水浴。使用钳子的曲线，将钳子放在两个石蜡部分之间，然后轻轻地将截块分开。使用潮湿的画笔将分离的部分推到带正的粘附显微镜幻灯片上。首先，在60摄氏度的金属架上孵化滑梯20分钟，将石蜡熔化。然后根据文本协议，在室温下在补水列车中通过孵育将幻灯片去分离。将幻灯片存放在自来水中，以防止非特异性抗体结合。在此之后，在125摄氏度的柠檬酸盐缓冲液中孵育幻灯片30秒，90摄氏度孵化10秒。在用蒸馏水冲洗幻灯片五次之前，让滑梯冷却。接下来，在室温下将幻灯片孵育为0.3%过氧化氢20分钟。然后，用温和的搅拌将幻灯片洗涤五分钟，在洗涤缓冲液中三次。在将稀释原抗体的加湿室中孵育一夜，温度定在摄氏四度。在此之后，用轻轻的搅拌将幻灯片洗三次，五分钟。接下来，在室温下将稀释的生物素化二次抗体的幻灯片孵育一小时。用温和的搅拌将滑梯洗涤三次，在洗涤缓冲液中洗五分钟。然后，在黑暗中的室温下孵育在阿维丁-生物基化马萝卜过氧化物酶复合物中的幻灯片一小时。重复上述清洗。在此之后，用DAB过氧化氢基板品牌孵育幻灯片在室温下两到五分钟。用自来水冲洗幻灯片，将幻灯片浸入赤氧树脂溶液中，以对抗它们。然后用自来水彻底冲洗滑梯，直到水清澈。根据文本协议使幻灯片脱水。最后，用二甲苯基安装介质盖住幻灯片，并在室温下将它们干燥在平坦的表面上。在此协议中，NS 是固定的，并嵌入在红糖和石蜡中。然后，他们被染色为特定的胶质瘤生物标志物。ATRX是一种调解染色质结构的伴郎蛋白，其表达水平较低。Ki67是细胞增殖的生物标志物，在胶质瘤NS中呈阳性。最后，NS对OLI62也呈阳性，这是一种促进寡头细胞分化的转录因子。要记住的最重要的事情是将细胞重新悬浮在温暖的阿加罗斯中，然后再转移到冰中，以确保神经球的均匀分布。

Research

JoVE Journal

Cancer Research

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

一种内在的弥漫性脑胶质瘤脑桥快速验尸细胞培养协议（DIPG）

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

科研

癌症研究

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

档案福尔马林固定，石蜡包埋的人类口咽鳞状细胞癌样品中T细胞亚群的四色荧光免疫组织化学

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

Determination of Protein Expression Level in Cultured Cells by Immunocytochemistry on Paraffin-embedded Cell Blocks

石蜡嵌段细胞免疫细胞化学法测定培养体细胞蛋白质表达水平

Immunofluorescent staining is currently the method of choice for determination of protein expression levels in cell-culture systems when morphological ...

Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue

pT3N0M0 胃癌组织石蜡嵌入幻灯片上的弹性染色

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

评估癌细胞3D球形培养中的细胞活力和死亡

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures

多端根结核细胞迁移的实时监测

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

快速筛选工作流程，利用患者衍生胶质瘤干细胞确定GBM的潜在组合疗法

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...

Co-culture of Glutamatergic Neurons and Pediatric High-Grade Glioma Cells Into Microfluidic Devices to Assess Electrical Interactions

将谷氨酸能神经元和儿科高级别胶质瘤细胞共培养到微流体装置中以评估电相互作用

Pediatric high-grade gliomas (pHGG) represent childhood and adolescent brain cancers that carry a rapid dismal prognosis. Since there is a need to ...

Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma

小儿弥漫性中线胶质瘤的活-3D细胞免疫细胞化学测定

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using ...

Digital Spatial Profiling for Characterization of the Microenvironment in Adult-Type Diffusely Infiltrating Glioma

用于表征成人型弥漫性浸润性胶质瘤微环境的数字空间剖析

Diffusely infiltrating gliomas are associated with high morbidity and mortality due to the infiltrative nature of tumor spread. They are morphologically ...