Name: simple detection of primary cilia by immunofluorescence
Uploaded: 2020-05-15T14:00:00
Description: Watch this Scientific Journal Video about Simple Detection of Primary Cilia by Immunofluorescence at JoVE.com

这项工作得到了捷克共和国国防部的支持 - 国防大学军事卫生科学系大规模毁灭性武器的长期组织发展计划医疗方面;捷克共和国教育、青年和体育部（具体研究项目号：SV/FVZ201703）和PROGRES Q40/06。也感谢丹尼尔·迪亚兹在英语修订方面提供的那种帮助。

1. 文化媒体、解决方案和菜肴的准备
<ol><li>自动保存盖玻片（22 x 22 mm）。准备6个井板。解冻胎儿牛血清（FBS）和抗生素青霉素/链霉素，并加热培养中到室温（RT）。使用 trypsin-EDTA （0.25%）和1x PBS（磷酸盐缓冲盐水与钙和镁）通过细胞。</li>
	<li>在 dH 2 O 中准备新鲜 4% 的甲状甲醛 （PFA）（20mL 的 dH2O 中为 800 毫克的 PFA）。PFA 必须为每个实验进行新鲜准备。在 55°C 下搅拌并加热溶...

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+levels+of+cytosolic+tubulin+regulate+ciliary+length+control.">Soluble levels of cytosolic tubulin regulate ciliary length control.</a> Molecular Biology of the Cell. 22 (6), 806-816 (2011).</li><li>Filipov&#225;, 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=Ionizing+radiation+increases+primary+cilia+incidence+and+induces+multiciliation+in+C2C12+myoblasts.">Ionizing radiation increases primary cilia incidence and induces multiciliation in C2C12 myoblasts.</a> Cell Biology International. 39 (8), 943-953 (2015).</li><li>Filipov&#225;, 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=The+toxic+effect+of+cytostatics+on+primary+cilia+frequency+and+multiciliation.">The toxic effect of cytostatics on primary cilia frequency and multiciliation.</a> Journal of Cellular and Molecular Medicine. 23 (8), 5728-5736 (2019).</li><li>Gadadhar, 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=Tubulin+glycylation+controls+primary+cilia+length.">Tubulin glycylation controls primary cilia length.</a> The Journal of Cell Biology. 216 (9), 2701-2713 (2017).</li><li>Shamloo, 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=Chronic+Hypobaric+Hypoxia+Modulates+Primary+Cilia+Differently+in+Adult+and+Fetal+Ovine+Kidneys.">Chronic Hypobaric Hypoxia Modulates Primary Cilia Differently in Adult and Fetal Ovine Kidneys.</a> Frontiers in Physiology. 8, 677 (2017).</li><li>Malone, A. M. D., 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=Primary+cilia+mediate+mechanosensing+in+bone+cells+by+a+calcium-independent+mechanism.">Primary cilia mediate mechanosensing in bone cells by a calcium-independent mechanism.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (33), 13325-13330 (2007).</li><li>Luesma, M. 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=Enteric+neurons+show+a+primary+cilium.">Enteric neurons show a primary cilium.</a> Journal of Cellular and Molecular Medicine. 17 (1), 147-153 (2013).</li><li>Pugacheva, E. N., Jablonski, S. A., Hartman, T. R., Henske, E. P., Golemis, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HEF1-dependent+Aurora+A+activation+induces+disassembly+of+the+primary+cilium.">HEF1-dependent Aurora A activation induces disassembly of the primary cilium.</a> Cell. 129 (7), 1351-1363 (2007).</li><li>Li, 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=Ciliary+transition+zone+activation+of+phosphorylated+Tctex-1+controls+ciliary+resorption,+S-phase+entry+and+fate+of+neural+progenitors.">Ciliary transition zone activation of phosphorylated Tctex-1 controls ciliary resorption, S-phase entry and fate of neural progenitors.</a> Nature Cell Biology. 13 (4), 402-411 (2011).</li><li>Spalluto, C., Wilson, D. I., Hearn, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+reciliation+of+RPE1+cells+in+late+G1+phase,+and+ciliary+localisation+of+cyclin+B1.">Evidence for reciliation of RPE1 cells in late G1 phase, and ciliary localisation of cyclin B1.</a> FEBS Open Bio. 3, 334-340 (2013).</li><li>Malicki, J. J., Johnson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cilium:+Cellular+Antenna+and+Central+Processing+Unit.">The Cilium: Cellular Antenna and Central Processing Unit.</a> Trends in Cell Biology. 27 (2), 126-140 (2017).</li><li>Morleo, M., Franco, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Autophagy-Cilia+Axis:+An+Intricate+Relationship.">The Autophagy-Cilia Axis: An Intricate Relationship.</a> Cells. 8 (8), 905 (2019).</li><li>Ott, C., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+live+primary+cilia+dynamics+using+fluorescence+microscopy.">Visualization of live primary cilia dynamics using fluorescence microscopy.</a> Current Protocols in Cell Biology. , (2012).</li><li>Sun, S., Fisher, R. L., Bowser, S. S., Pentecost, B. T., Sui, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+architecture+of+epithelial+primary+cilia.">Three-dimensional architecture of epithelial primary cilia.</a> Proceedings of the National Academy of Sciences. 116 (19), 9370-9379 (2019).</li><li>Lauring, M. 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=New+software+for+automated+cilia+detection+in+cells+(ACDC).">New software for automated cilia detection in cells (ACDC).</a> Cilia. 8 (1), 1 (2019).</li><li>Conroy, P. 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=C-NAP1+and+rootletin+restrain+DNA+damage-induced+centriole+splitting+and+facilitate+ciliogenesis.">C-NAP1 and rootletin restrain DNA damage-induced centriole splitting and facilitate ciliogenesis.</a> Cell Cycle. 11 (20), 3769-3778 (2012).</li><li>Kim, J. 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=Genome-wide+screen+identifies+novel+machineries+required+for+both+ciliogenesis+and+cell+cycle+arrest+upon+serum+starvation.">Genome-wide screen identifies novel machineries required for both ciliogenesis and cell cycle arrest upon serum starvation.</a> Biochimica et Biophysica Acta. 1863 (6), 1307-1318 (2016).</li><li>Yuan, 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=Primary+cilia+are+decreased+in+breast+cancer:+analysis+of+a+collection+of+human+breast+cancer+cell+lines+and+tissues.">Primary cilia are decreased in breast cancer: analysis of a collection of human breast cancer cell lines and tissues.</a> The Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society. 58 (10), 857-870 (2010).</li><li>Smith, 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=Differential+HDAC6+Activity+Modulates+Ciliogenesis+and+Subsequent+Mechanosensing+of+Endothelial+Cells+Derived+from+Pluripotent+Stem+Cells.">Differential HDAC6 Activity Modulates Ciliogenesis and Subsequent Mechanosensing of Endothelial Cells Derived from Pluripotent Stem Cells.</a> Cell Reports. 24 (4), 895-908 (2018).</li><li>Mirvis, M., Siemers, K. 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几位,作者描述了检测原发性西莉亚的不同方法，有时也描述了可能影响其检测6、20、21、2220,21的各种固定方法。6无论如何，很难找到一个完整而简单的检测协议。这种方法的可得性无疑对研究原发性西莉亚研究大有帮助，特别是在研究的早期阶段，或对测试所选细胞系中是否存在原发性西莉亚的迅?...

原发性胆汁是感觉，孤独，膜绑定，非mo动结构与细胞的母亲中心。除红血球、脂肪细胞1和肝细胞2外，大多数脊椎动物细胞都发现了原发性细胞。原发性西莉亚形成为由微管组成的拉长轴酮，其主要成分为β-管状素。斧头从基底体生长，基底体由+-tubulin组成。主西莉亚的长度在2~10μm之间变化;,然而，在糖化、饥饿、缺氧、细胞毒性应激或暴露于电离辐射3、4、5、6、7,4之后，5,6其尺寸可能会发生变化。通常，细胞只有一个原发性，它涉及形态生成和细胞信号通路对细胞增殖和分化88，9至关重要。
原发性cilia在细胞周期进展期间，特别是在G0/G1阶段，在与HDAC6（组蛋白去乙酰酶6）调解的细胞脱乙酰化相关过程中，在进入线粒体之前重新生长。原发性西莉亚吸收的确切时刻取决于细胞类型和直接参与这个过程的基因的表达，如极光A、Plk1、TcTex-111、12、13。11,12,13根据细胞类型，原发性cilia表达不同类型的受体、离子通道和有源信号通路。其中包括影响增殖和生存的最重要的信号受体、EGFR、PDGFR 和 FGFR。还包括一些可能影响一个或多个器官功能的信号通路，包括刺猪、诺奇和Wnt。 由于这些受体和信号通路，主要细胞也执行化学感觉功能。此功能允许原发性西莉亚检测诺奇、激素和生物活性物质（如血清素或生长激素）的特定配体。不同长度的原发性西莉亚所表现出的其他特定功能包括对温度、重力和渗透性14的变化的反应。
原发cilia可以通过各种方法进行可视化，如实时可视化、传输电子显微镜、3D成像，或通过软件自动检测原发cilia5、15、16、17。5,15,16,17然而，这些方法是高度专业化和持续的研究需要基本，快速，简单的方法染色原发性西莉亚在研究的每个阶段。描述是一种简单而有用的方法，用于检测培养细胞中的原发性西莉亚。

<table border="0" cellpadding="0" cellspacing="0" style="width:1301px;" width="1301">
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			<td height="21" style="height:21px;width:384px;">6-well plate</td>
			<td style="width:217px;">TPP</td>
			<td style="width:119px;">92406</td>
			<td style="width:581px;">Dimensions 128x86x22 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Alexa Fluor488</td>
			<td style="width:217px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">111-546-047</td>
			<td>AffiniPure F(ab&#39;)&#8322; Fragment Goat Anti-Rabbit IgG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Anti-Tubulin &#947;</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">T5192</td>
			<td>Polyclonal Rabbit anti-Mouse IgG2a</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">C2C12</td>
			<td style="width:217px;">ATCC</td>
			<td style="width:119px;">CRL-1772</td>
			<td>Myoblast (mouse)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Cy3</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">C2181</td>
			<td>Anti-Mouse IgG (whole molecule) F(ab&#8242;)2 fragment&#8211;Cy3 antibody produced in sheep</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Dapi (4&#8242;,6-Diamidino-2-phenylindole dihydrochloride)</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Dulbecco&#180;s Modified Eagle&#180;s medium</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:119px;">11960044</td>
			<td>High glucose, No glutamine, Gibco</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:384px;">Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8662</td>
			<td>With MgCl2 and CaCl2, Sterile-filtered, Suitable for cell culture</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Fetal Bovine Serum</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:119px;">16000044</td>
			<td>Sterile-Filtered, Gibco</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:384px;">L-Glutamine</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">G7513</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:384px;">MEF</td>
			<td style="width:217px;">ATCC</td>
			<td style="width:119px;">SCRC-1039</td>
			<td>Mouse embryonic fibroblast</td>
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			<td height="21" style="height:21px;width:384px;">Monoclonal Anti-Acetylated Tubulin</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">T7451</td>
			<td>Monoclonal Anti-Acetylated Tubulin antibody produced in mouse</td>
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			<td height="21" style="height:21px;width:384px;">NHLF</td>
			<td style="width:217px;">Lonza</td>
			<td style="width:119px;">CC-2512</td>
			<td>Primary lung fibroblasts (human)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Normal Goat Serum</td>
			<td style="width:217px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">005-000-121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Paraformaldehyde</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">158127-500G</td>
			<td>Powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Penicillin-Streptomycin</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">P0781</td>
			<td>10,000 units penicillin and 10 mg streptomycin per mL in 0.9% NaCl, Sterile-Filtered</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">ProLong Diamond Antifade Mountant</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:119px;">P36961</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:384px;">Skin fibroblasts</td>
			<td style="width:217px;">Kindly gifted from Charles University, Faculty of Medicine in Hradec Kr&#225;lov&#233;.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Square Cover Slips</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:119px;">22X22-1.5</td>
			<td>Borosilicate glass, 22x22mm, Square</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Triton X-100</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">11332481001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Trypsin-EDTA (0.25%)</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:119px;">25200072</td>
			<td>Sterile-Filtered, Gibco</td>
		</tr>
	</tbody>
</table>

simple detection of primary cilia by immunofluorescence

原生性细胞在细胞周期进展期间动态调节，特别是在细胞周期的G0/G1阶段，在分粒之前被重新吸收。通过高度复杂的方法（包括传输电子显微镜、3D 成像或使用软件自动检测原发cilia）可以可视化原发cilia。然而，需要免疫荧光染色原发性西莉亚执行这些方法。本出版物描述了通过染色乙酰化α管状图布图林（axoneme）和伽马图布林（基础体）在体外轻松检测原发性西莉亚的协议。这种免疫荧光染色方案相对简单，可产生高质量的图像。本协议描述了表达原发性西莉亚的四个细胞系（C2C12、MEF、NHLF和皮肤成纤维细胞）如何固定、免疫，以及如何用荧光或共体显微镜成像。

原发性西莉亚的免疫荧光染色是一个相对简单的过程，可以产生高质量的图像。在这些实验中，表达原发性西莉亚的成纤维细胞按照上述协议在荧光或共合显微镜中固定、免疫和成像。使用乙酰β-图布林和β-图布林检测出原发性柠檬。原发性西莉亚的评价可以在不同级别上进行，这方面的任何变化都可以与电离辐射、细胞代谢（如饥饿）或化学处理（如细胞静电）5、18,<...

Watch this Scientific Journal Video about Simple Detection of Primary Cilia by Immunofluorescence at JoVE.com

Simple Detection of Primary Cilia by Immunofluorescence

原发性西莉亚是与中环相关的细胞外结构。通过免疫荧光染色进行初级西莉亚检测是一个相对简单的过程，可产生极高质量的图像。在该协议中，表达原发性西莉亚的成纤维细胞被固定、免疫化，并在荧光或共合显微镜中成像。

通过免疫荧光简单检测原发性西莉亚

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to ...

该协议允许在各种研究努力中快速轻松地检测培养细胞中的原发性西莉亚。该方法可用于影响原发性西莉亚基体的混乱。此过程还可用于在石蜡嵌入组织中染色原发性西莉亚，或用于需要荧光的其他实验。首先，用高压灭22毫米盖滑，为实验准备材料。解冻FBS和青霉素链霉素，并温暖培养中室温度。通过带三辛EDTA和PBS的细胞。根据手稿说明，准备新鲜的 4% 甲醛、培养基和 1% 明胶溶液。然后用70%乙醇清洁层流罩，并放置所有必需的材料。甲醛是极其危险的。必须时刻佩戴适当的 PPE，并且只能在化学罩中处理。将所需的细胞生长到70%汇合后，将它们从培养箱中取出，并放在层流罩中。取出培养介质，用 PBS 冲洗细胞两次。在培养瓶中加入约两毫升0.25%的三丁辛EDTA，并在37摄氏度下孵育5分钟。定期在倒置显微镜上检查细胞，以监测分离。当细胞分离时，在10毫升培养培养中轻轻重新悬浮，小心移液，形成单个细胞悬浮液。将电池悬浮液转移到50毫升锥形管中，并在200次G'下离心5分钟。去上一液，然后加入10毫升的培养介质，轻轻重新暂停颗粒。服用20微升的细胞悬浮液，并混合与尝试蓝在一对一的体积比。然后使用标准血细胞切除术计算细胞。将盖滑放在六井板的每个井内，用明胶溶液的两毫升涂覆滑，这将有助于附着在盖板上的细胞。取出明胶，让板风干几分钟。种子10万成纤维细胞进入每井，并添加两毫升的培养媒介。然后在37摄氏度、5%的二氧化碳和90%的相对湿度下孵育细胞24小时。孵育后治疗细胞诱导细胞。将 4%的甲状甲醛加热至室温，并准备牧场移液器、ABS、废容器、15 毫升锥形管、微型移液器和尖端。从培养箱中取出细胞，放在实验室的长凳上。从每个井上拆下介质，使盖滑。用两毫升 PBS 轻轻清洗细胞三次。然后将 4%PFA 的两毫升添加到每个井中以修复细胞。在室温下孵育板10分钟，然后取出PFA，然后用PBS重复洗涤。在每个井中加入两毫升0.5%Triton X-100，并孵育板15分钟。然后用PBS洗四次井。要制作阻断溶液，解冻山羊血清五分钟，并在PBS中以1比20的比例稀释。将溶液的150微升添加到每个盖滑中，并孵育板20分钟。解冻原抗体五分钟，并在PBS中单独稀释，如文本手稿中所述。从盖滑道上去除阻抗溶液，并添加两种抗体溶液的 150 微升。然后在室温下孵育板60分钟。取出原抗体，用两毫升PBS轻轻清洗盖滑三次。在PBS中稀释Cy3绵羊抗小鼠和Alexa氟488山羊抗兔，比例为1比300，并在盖滑中加入150微升的二次抗体溶液。在 50 毫升 PBS 中稀释 10 微升库存，并准备工作 DAPI 解决方案，并将稀释的 2 毫升添加到盖滑中。在黑暗中，在室温下孵育板5分钟。在将盖板滑在幻灯片上之前，请准备两根针、钳子和安装介质，并标记所有幻灯片。从井中去除 DAPI 溶液，用 PBS 清洗三次。在每张幻灯片上放置一滴安装介质。使用针头轻轻地将盖滑从井底提起。然后翻转它，轻轻地把它放在安装介质的滴使用钳子。小心地清除任何气泡。保护幻灯片免受光线影响，并在四摄氏度下过夜。第二天，使用具有高放大率的荧光或共和显微镜来可视化原发性西莉亚。该协议用于修复免疫污，并图像各种细胞系表达原发性cilia。当小鼠肌细胞以各种剂量暴露在电离辐射下时，发现高剂量会诱发多种原发性西莉亚的出现。而低剂量导致单一原发性西莉亚的出现。同样，当人类肺成纤维细胞以低剂量辐射时，通过免疫荧光检测到单一原发性丝细胞。在饥饿的小鼠胚胎成纤维细胞中观察到原发性丝丝的增加，这表明代谢应激影响原发性西莉亚的事故。当皮肤成纤维细胞被120纳米摩尔多索鲁比辛治疗时，它们表示一个单一原发性柠檬。高剂量诱导多个原发性西莉亚的外观。用1.25纳米摩尔分类治疗的成纤维细胞也表达单一原发性。但治疗剂量较高后未检测到多个西莉亚。打开此协议时，请务必遵守所有 PPE 法规。请记住您选择的细胞系的培养需求，并仔细选择您的抗体。此过程不能直接遵循其他程序，因为单元格是不可恢复的。相反，应对并行实验进行编程。

Research

JoVE Journal

Biology

Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue

Immunofluorescent Detection of Two Thymidine Analogues CldU and IdU in Primary Tissue

小学组织两个胸苷类似物的免疫荧光检测（CldU IDU）

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are ...

科研

生物学

Detection of Functional Matrix Metalloproteinases by Zymography

酶谱法检测功能的基质金属蛋白酶

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

检测采用ELISA法的母语完整的核小体的翻译后修饰

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

荧光检测病毒RNA<em&gt;原位</em&gt;杂交（FISH）

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

基因功能分析和可视化的纤毛产生的流体流动的在枯否囊泡中

Internal organs such as the  heart, brain, and gut develop left-right (LR) asymmetries that are critical for  their normal functions1. Motile cilia are ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

检测现场的<em&gt;大肠杆菌</em&gt; O157：H7细胞的PMA-qPCR的

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

糖皮质激素粪分析：在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

配体激活的G蛋白偶联受体内在化的检测通过共聚焦显微镜

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...