Name: fluorescent immunolocalization of arabinogalactan proteins and pectins in the cell wall of plant tissues
Uploaded: 2021-02-27T13:00:00
Description: Watch this Scientific Journal Video about Fluorescent Immunolocalization of Arabinogalactan Proteins and Pectins in the Cell Wall of Plant Tissues at JoVE.com

作者得到了由H2020-MSCA-RISE-2015和种子轮FCT PTDC/BIA-FBT/27839/2017资助的欧盟项目690946"性种子"（性植物繁殖-种子形成）的支持。AMP获得了欧盟MSCA-IF-2016项目（第753328号）的赠款。MC 获得了 FCT 博士资助 SFRH/BD/111781/2015 的资助。

1. 样品准备：固定、脱水和LR-白色嵌入
<ol><li>固定和脱水 注：固定过程对于保存样本至关重要：通过交叉链接分子，细胞代谢停止，确保细胞完整性，防止分子扩散。固定剂和使用的浓度必须调整到该目的，使抗原充分暴露与抗体相互作用。以下协议结合了准甲醛的轻度固定能力，以及谷胱甘肽的更强效果。其比例被优化为AGP和细胞壁组件，但它适用于其他蛋白质和细胞结构。?...

<ol>
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L., Coimbra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabinogalactan+Proteins+as+Interactors+along+the+Crosstalk+between+the+Pollen+Tube+and+the+Female+Tissues.">Arabinogalactan Proteins as Interactors along the Crosstalk between the Pollen Tube and the Female Tissues.</a> Frontiers in Plant Science. 7 (7), (2016).</li><li>Showalter, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabinogalactan-proteins:+structure,+expression+and+function.">Arabinogalactan-proteins: structure, expression and function.</a> Cellular and Molecular Life Sciences. 58 (10), 1399-1417 (2001).</li><li>Majewska-Sawka, A., Nothnagel, 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+multiple+roles+of+arabinogalactan+proteins+in+plant+development.">The multiple roles of arabinogalactan proteins in plant development.</a> Plant Physiology. 122, 3-9 (2000).</li><li>Seifert, G., Roberts, 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+biology+of+arabinogalactan+proteins.">The biology of arabinogalactan proteins.</a> Annual Review of Plant Biology. 58, 137-161 (2007).</li><li>Ruprecht, 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=A+Synthetic+Glycan+Microarray+Enables+Epitope+Mapping+of+Plant+Cell+Wall+Glycan-Directed+Antibodies.">A Synthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies.</a> Plant Physiology. 175 (3), 1094-1104 (2017).</li><li>Verhertbruggen, Y., Walker, J. L., Guillon, F., Scheller, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comparative+Study+of+Sample+Preparation+for+Staining+and+Immunodetection+of+Plant+Cell+Walls+by+Light+Microscopy.">A Comparative Study of Sample Preparation for Staining and Immunodetection of Plant Cell Walls by Light Microscopy.</a> Frontiers in Plant Science. 29 (8), 1505 (2017).</li><li>Osborn, M., Weber, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+and+immunocytochemical+procedures+with+affinity+purified+antibodies:+tubulin+containing+structures.">Immunofluorescence and immunocytochemical procedures with affinity purified antibodies: tubulin containing structures.</a> Methods in Cell Biology. 24, 97-132 (1982).</li><li>Coimbra, S., Almeida, J., Junqueira, V., Costa, M., Pereira, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabinogalactan+proteins+as+molecular+markers+in+Arabidopsis+thaliana+sexual+reproduction.">Arabinogalactan proteins as molecular markers in Arabidopsis thaliana sexual reproduction.</a> Journal of Experimental Botany. 58 (15), 4027-4035 (2007).</li><li>Wilson, S. M., Bacic, A. <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+plant+cells+for+transmission+electron+microscopy+to+optimize+immunogold+labeling+of+carbohydrate+and+protein+epitopes.">Preparation of plant cells for transmission electron microscopy to optimize immunogold labeling of carbohydrate and protein epitopes.</a> Nature Protocols. 7 (9), 1716-1727 (2012).</li><li>Gane, A., Clarke, A., Bacic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+and+expression+of+arabinogalactan-proteins+in+the+ovaries+of+Nicotiana+alata+Link+and+Otto.">Localization and expression of arabinogalactan-proteins in the ovaries of Nicotiana alata Link and Otto.</a> Sex Plant Reproduction. 8, 278-282 (1995).</li><li>Coimbra, S., Salema, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunolocalization+of+arabinogalactan+proteins+in+Amaranthus+hypocondriacus+L.+ovules.">Immunolocalization of arabinogalactan proteins in Amaranthus hypocondriacus L. ovules.</a> Protoplasma. 199 (1-2), 75-82 (1997).</li><li>Coimbra, S., Duarte, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabinogalactan+proteins+may+facilitate+the+movement+of+pollen+tubes+from+the+stigma+to+the+ovules+in+Actinidia+deliciosa+and+Amaranthus+hypocondriacus.">Arabinogalactan proteins may facilitate the movement of pollen tubes from the stigma to the ovules in Actinidia deliciosa and Amaranthus hypocondriacus.</a> Euphytica. 133 (2), 171-178 (2003).</li><li>El-Tantawy, 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=Arabinogalactan+protein+profiles+and+distribution+patterns+during+microspore+embryogenesis+and+pollen+development+in+Brassica+napus.">Arabinogalactan protein profiles and distribution patterns during microspore embryogenesis and pollen development in Brassica napus.</a> Plant Reproduction. 26 (3), 231-243 (2013).</li><li>Costa, M., Pereira, A. M., Rudall, P. J., Coimbra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunolocalization+of+arabinogalactan+proteins+(AGPs)+in+reproductive+structures+of+an+early-divergent+angiosperm,+Trithuria+submersa+(Hydatellaceae).">Immunolocalization of arabinogalactan proteins (AGPs) in reproductive structures of an early-divergent angiosperm, Trithuria submersa (Hydatellaceae).</a> Annals of Botany. 111 (2), 183-190 (2013).</li><li>Costa, M., Sobral, R., Ribeiro Costa, M. M., Amorim, M. I., Coimbra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+presence+of+arabinogalactan+proteins+and+pectins+during+Quercus+suber+male+gametogenesis.">Evaluation of the presence of arabinogalactan proteins and pectins during Quercus suber male gametogenesis.</a> Annals of Botany. 115 (1), 81-92 (2015).</li><li>Hibbs, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+Immunolabelling.">Fluorescence Immunolabelling.</a> Confocal Microscopy for Biologists. , (2004).</li><li>Johnson, G. 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=Fading+of+Immunofluorescence+during+microscopy:+A+Study+of+the+Phenomenon+and+its+Remedy.">Fading of Immunofluorescence during microscopy: A Study of the Phenomenon and its Remedy.</a> Journal of Immunological Methods. 55 (2), 231-242 (1982).</li><li>Baird, T. R., Kaufman, D., Brown, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mercury+Free+Microscopy:+An+Opportunity+for+Core+Facility+Directors.">Mercury Free Microscopy: An Opportunity for Core Facility Directors.</a> Journal of Biomolecular Techniques. 25 (2), 48-53 (2014).</li><li>Weber, G. F., Menko, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Color+image+acquisition+using+a+monochrome+camera+and+standard+fluorescence+filter+cubes.">Color image acquisition using a monochrome camera and standard fluorescence filter cubes.</a> BioTechniques. 38 (1), 52-56 (2005).</li></ol>

这里描述的植物荧光免疫化方法虽然无缝简单，但依赖于几个小步骤的成功。第一个是样品准备和固定。在第一步中，甲醛和谷胱甘肽的混合物用于交叉连接大多数细胞组件。溶液中的甲醛提供温和和可逆的固定，而谷胱甘肽提供更强的永久联系：两个固定剂之间的平衡提供了适当的交叉链接量，允许暴露的表位与主要抗体10反应。要使固定解决方案正常工作，样本必须很小。?...

植物细胞壁是由多糖和糖蛋白组成的复杂结构。细胞壁是极具活力的结构，其结构、组织和组成因细胞类型、定位、发育阶段、外部和内部刺激1而异。阿拉比诺格拉坦蛋白（AGPs）和皮丁是植物细胞壁的重要组成部分。AGP是高糖化蛋白，果胶是同源多糖，其组成、数量和结构在不同的植物发育阶段2、3、4差异很大。AGP和果胶研究表明，他们参与了几个植物过程，如编程细胞死亡，对副生压力的反应，性植物繁殖，等等5。这些研究大多从免疫化研究获得的信息开始。
鉴于其复杂性，研究细胞壁需要许多不同的工具。使用单克隆抗体 （mAbs） 检测甘草表位是解决聚糖和糖蛋白沿此结构分布的宝贵方法。有大量的mAb可用于检测甘油表位和每个mAb的特异性正在不断改善，以及6。此处描述的技术适用于所有植物物种，是指导未来研究方向的完美工具，可能涉及更昂贵和复杂的技术。
在这项技术中，特定的抗体在化学上与荧光染料结合，如FITC（荧光异硫氰酸酯）、TRITC（四甲基多胺-5-（和6）异硫氰酸酯）或几种亚历克萨氟酸盐染料。免疫荧光提供了几个优点，允许在荧光显微镜下直接观察到的甘精的清晰和快速的亚细胞定位。它是高度具体和敏感的，因为样品的制备可以有效地保护抗原的自然结构，即使存在于较低的量。它允许检测多个抗原在同一样本中，最重要的是，提供高质量和视觉上美丽的结果。尽管荧光免疫研究提供了巨大的力量，但它们往往被认为难以执行和实施，很可能是因为缺乏允许可视化程序不同步骤的详细方案。在这里，我们提供了一些简单的指南，如何执行这项技术，以及如何获得高质量的图像。
对于此处提交的协议，样品必须首先使用最合适的固定剂进行固定和嵌入。虽然被认为是一种耗时且相对繁琐的技术，但植物样本的正确固定和嵌入是确保免疫化检测成功的关键。为此，最常见的是使用交叉链接固定剂（如醛）进行化学固定。交叉连接固定剂在组织分子之间建立化学键，稳定和硬化样品。甲醛和谷胱甘肽是交叉连接固定剂，有时两种固定剂的混合物使用7。甲醛为组织提供了很好的结构保存和长时间的保存，产生小组织缩水，并与免疫污染相容。谷胱甘肽是一种更强、更稳定的固定剂，通常与甲醛结合使用。谷胱甘肽的使用有一些缺点，必须考虑到，因为它引入一些自由醛组到固定组织，这可能会产生一些不具体的标签。此外，蛋白质和其他分子之间的交叉连接偶尔可能会使一些目标表位无法接触到抗体。为了避免这种情况，必须仔细定义固定的数量和持续时间。
固定后，样品嵌入适当的树脂中，在获得部分之前变硬。伦敦树脂（LR-White）丙烯酸树脂是免疫化研究的首选树脂。与其他树脂不同，LR-White是亲水的，允许抗体达到其抗原，无需任何治疗，以促进它。LR-White 还具有提供低自动荧光的优势，允许在免疫荧光成像过程中降低背景噪音。
有许多染色技术可用于检测细胞壁的不同成分，如阿尔西亚蓝色染色，甲丁蓝色染色或周期性酸-希夫（PAS）染色。这些都没有提供免疫分析的力量8。这种方法在糖的检测方面具有更大的特异性，提供了关于细胞壁组成和结构的更广泛的信息。

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">25% (w/v) Gluteraldehyde</td>
			<td style="width:99px;">Agar Scientific</td>
			<td style="width:196px;">AGR1010</td>
			<td style="width:187px;">aq. Solution, methanol free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">8 wells Glass reaction slides</td>
			<td style="width:99px;">Marinfeld</td>
			<td style="width:196px;">MARI1216750</td>
			<td style="width:187px;">other brands may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Acetic acid</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:196px;">A6283</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Anti-Rat IgG (wole molecule)-FITC antibody produce in GOAT</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:196px;">F6258</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:247px;">cover slips, 24 mm x 50 mm</td>
			<td style="width:99px;">Marinfeld</td>
			<td>MARI0100222</td>
			<td style="width:187px;">The cover slip should cover all the wells. Other brands may be used&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:247px;">ddH2O</td>
			<td style="width:99px;">na</td>
			<td style="width:196px;">na</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Ethanol absolute</td>
			<td style="width:99px;">na</td>
			<td style="width:196px;">na</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Fluorescent brightner 28</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:196px;">F-6259</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Gelatin capsules</td>
			<td style="width:99px;">Agar scientific</td>
			<td>AGG29211</td>
			<td style="width:187px;">The capsule size sould feat the size of the sample.</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:247px;">Glass vials</td>
			<td style="width:99px;">na</td>
			<td style="width:196px;">na</td>
			<td style="width:187px;">Any simple unexpensive glass vials that can be sealed, may be used. The vials may be clean with 96% etanol after use to remove LR-White residue and reused.&#160;</td>
		</tr>
		<tr height="294">
			<td height="294" style="height:294px;width:247px;">LR-white medium grade, embdeding resin</td>
			<td style="width:99px;">Agar Scientific</td>
			<td>AGR1281</td>
			<td style="width:187px;">LR-White comes in several forms the medium grade provides na adequate cutting suport for most tissues for harder tissues a harder grade of LR-white may be recomendable. If possible use a resin already mixed with the polimeration activator (benzoyl peroxide), if not please folow the instructions of the suplier to prepare the resin.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Non fat dry milk</td>
			<td style="width:99px;">Nestl&#233;</td>
			<td style="width:196px;">na</td>
			<td style="width:187px;">any non fat dry milk is adequate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Oven</td>
			<td style="width:99px;">na</td>
			<td style="width:196px;">na</td>
			<td style="width:187px;">generic laboratory oven</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Petri dish, 10 cm x 10 cm square</td>
			<td style="width:99px;">na</td>
			<td style="width:196px;">na</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PIPES</td>
			<td style="width:99px;">Sigma Aldrich</td>
			<td style="width:196px;">P1851</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="357">
			<td height="357" style="height:357px;width:247px;">Rat generated Monoclonal Anti-Body</td>
			<td style="width:99px;">Plant probes</td>
			<td style="width:196px;">na</td>
			<td style="width:187px;">Several antibodies that recognize cell wall components are 
			available at both the Complex Carbohydrate research center 
			(CCRC, Georgia University USA) and Plant Probes (Paul 
			Knox Cell Wall Lab, at Leeds University UK). A short list of 
			some commonly used MABS and where they can be purchased 
			is presented in Supplemental Table 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Razor blades</td>
			<td style="width:99px;">na</td>
			<td style="width:196px;">na</td>
			<td style="width:187px;">regular razor blades</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">SDS</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:196px;">L6026</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Toluidine Blue-O</td>
			<td style="width:99px;">Agar Scientific</td>
			<td>AGR1727</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Tween 20</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:196px;">P9416</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Ultramicrotome</td>
			<td style="width:99px;">Leica Microsystems</td>
			<td style="width:196px;">UC7</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:247px;">upright epifluorescence microscope with UV and FITC fluorescence filters</td>
			<td style="width:99px;">Leica Mycrosistems</td>
			<td style="width:196px;">DMLb</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">vaccum chamber</td>
			<td style="width:99px;">na</td>
			<td style="width:196px;">na</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">vaccum pump</td>
			<td style="width:99px;">na</td>
			<td style="width:196px;">na</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="297">
			<td height="297" style="height:297px;width:247px;">Vectashield</td>
			<td style="width:99px;">vecta Labs</td>
			<td style="width:196px;">T-1000</td>
			<td style="width:187px;">Other anti-fade may be used. Please do check for compatibility with FITC and the Fluorescente brightner 28. (Note: for a non-commercial alternative, (see Jonhson et al 198218) An antifade medium can be made by mixing 25 mg/mL of 1,4-Diazobicyclo-(2,2,2)octane (DABCO) in 9:1 (v/v) glycerol to 1xPBS. Adjust pH to 8.6 with diluted HCl.)</td>
		</tr>
	</tbody>
</table>

fluorescent immunolocalization of arabinogalactan proteins and pectins in the cell wall of plant tissues

植物发育需要根据内部和外部的刺激不断调整细胞壁的组成和结构。细胞壁由纤维素和非纤维素多糖以及蛋白质、酚类化合物和水组成。90%的细胞壁由多糖（例如皮茨坦）和阿拉伯甘草蛋白（AGPs）组成。植物组织学部分特定甘草表位的荧光免疫化仍然是揭示墙体多糖网络、结构和组件改造的关键工具。
在这里，我们报告一个优化的荧光免疫化程序，以检测来自AGP和植物组织中的皮瓣的甘油表位。与植物样品的LR-White嵌入一起使用甲醛/谷胱甘肽固定，从而更好地保存组织结构和组成。用超微切除术获得的嵌入式样品的细小部分用于特定抗体的免疫化。该技术提供高分辨率、高特异性以及检测同一样本中多个甘油表位的机会。该技术允许甘油的亚细胞定位，并检测其在细胞壁中的积累水平。它还允许在开发过程中确定AGP和果胶分布的空间-时间模式。使用此工具最终可能指导研究方向，并将甘草与植物中的特定功能联系起来。此外，获得的信息可以补充生化和基因表达研究。

在成功的实验中，次要抗体将以一致的方式以亮绿色具体定位特定表位的位置，从而在细胞、组织或器官的某一发育阶段对细胞壁组成进行定性。例如，LM6抗体对1，5-arabinan具有很高的亲和力，这种化合物具有I型拉诺拉图罗南，可以发现大量标记正在发育的 Quercus子 体的细胞壁（图2A），从而可以得出结论，这种类型的果胶是丰富的，是主要细胞壁成分的一部分。JIM5?...

Watch this Scientific Journal Video about Fluorescent Immunolocalization of Arabinogalactan Proteins and Pectins in the Cell Wall of Plant Tissues at JoVE.com

Fluorescent Immunolocalization of Arabinogalactan Proteins and Pectins in the Cell Wall of Plant Tissues

该协议详细描述了用于阿拉伯甘草蛋白和果胶免疫的植物材料是如何固定的，嵌入亲水丙烯酸树脂中，在玻璃滑梯上分节并安装。我们显示细胞壁相关的表位将被检测到与特定的抗体。

植物组织细胞壁中阿拉比诺格拉坦蛋白和皮克廷的荧光免疫化

Plant development involves constant adjustments of the cell wall composition and structure in response to both internal and external stimuli. Cell walls ...

只有一个实验的信息量是巨大的。我们可以检测这些细胞壁聚合物的存在，并确切地知道它们是如何分布在细胞和组织中的，甚至确定它们的丰度。这种方法的设计方式，它可以应用于几乎任何种类的植物组织从任何物种，人们可能想要分析。植物组织的免疫化涉及许多细致的工作，很难解释，没有实际看到会发生什么。在收集植物组织样本之前，在玻璃瓶中装满足够的冷固定溶液，以完全淹没所有样品。然后选择要分析的植物组织，并将样品修剪到不超过 16 平方毫米的大小。立即将样品浸入固定溶液中。谷胱甘肽和甲醛都是很好的固定剂，但两者都是危险的，必须在流罩中处理。将小瓶转移到真空室，慢慢吸尘。当压力达到负60公斤时，小瓶中的漂浮物将开始沉入底部。将室内压力保持在不超过负 80 千卡。下沉过程可能需要长达两个小时。样品在真空室中经过两个小时后，慢慢释放真空。密封玻璃瓶，在摄氏四度下冷藏过夜。隔夜固定后，丢弃任何没有沉入小瓶底部的样品。用25毫摩尔PBS清洗剩余样品10分钟，然后用25毫摩尔管道缓冲液清洗20分钟。删除所有浮动样品非常重要，因为固定样品极有可能没有穿透样品。将样品脱水至乙醇系列中，每次乙醇浓度浸入样品20分钟。为了渗透样品，在乙醇中加入越来越多的LR-白色树脂浓度，从一部分树脂开始到三部分乙醇。每次浓度，在4摄氏度下孵育24小时。用新鲜树脂代替LR-白色树脂，并在4摄氏度下将样品再孵育12小时。准备嵌入明胶胶囊，选择比样品稍大的胶囊，以便样品可以完全封闭在树脂中。用铅笔标记纸标签，因为墨水会污染树脂。将一滴新鲜的LR-白色树脂涂抹在每粒胶囊的底部。将样品放在每个胶囊中，然后用新鲜树脂填充胶囊，以达到最大容量。将盖子放在胶囊上，轻轻按压以密封。要将树脂聚合，在58摄氏度下固化胶囊，直到24至48小时左右完全硬化。从LR-白色树脂中去皮明胶胶囊后，将树脂块置于立体显微镜下。使用锋利的剃须刀刀片，以 45 度角修剪 LR-白色方块。旋转样品，以90度角向第一个切口进行第二次切割。继续以相同的模式切割，形成金字塔的顶点，直接位于样品感兴趣的区域之上，然后开始去除垂直于主轴的细片，直到切割表面到达样品。理想情况下，目标样品应封闭在梯形形状中。将修剪过的树脂块放置在超微切除术中。调整刀刃和树脂块之间的角度。从块上切割厚度在 200 到 700 纳米之间的部分。使用钢丝环小心地从微原子中提起一些采样部分。将它们放在玻璃滑梯上的一滴蒸馏去离子水中。将滑梯放在50摄氏度的热板上蒸发水，然后给部分添加一滴污渍。30 秒后，冲洗污渍并观察显微镜下的滑梯，以验证该部分的一般状态，并查看所需的植物结构。准备一个干净的反应幻灯片，在幻灯片的每口井中放置一滴蒸馏去离子水。将一到两个样本部分转移到每滴水中。将滑梯放在一个10厘米的方形培养皿中，盖上它，让它在50摄氏度下干燥。要为反应滑动准备一个孵化室，在移液器提示盒的底部放置一些阻尼纸巾，然后用铝箔包裹盒子。将幻灯片从盒子转移到孵化室。将 50 微升阻塞溶液放入每个井中。孵化幻灯片 10 分钟后，取出阻塞溶液，然后每次用 PBS 两次清洗所有油井 10 分钟。在准备了手稿中描述的主要抗体溶液后，用蒸馏除离子水对水井进行最后洗涤五分钟。将主要抗体溶液放入反应井中，然后将阻塞溶液移到控制井中。关闭孵化室。让它在室温下站立两个小时，然后在四摄氏度下冷藏过夜。在阻塞溶液中准备辅助抗体的 1% 溶液。每口井大约需要40微升。用铝箔盖住溶液。用PBS两次清洗反应滑梯的水井，每次用蒸馏的去离子水清洗一次，每次10分钟。确保井中不保留阻塞溶液或沉积物。将二次抗体溶液放入所有油井中。在室温下在黑暗中孵化滑梯三到四个小时。再次，用PBS洗两次反应滑梯的井，一次用蒸馏去离子水，然后给每口井加一滴白钙氟。无需清洗，在每个油井中加入一滴安装介质，并盖上每口油井的盖子。使用荧光显微镜观察反应幻灯片中的样本部分。对于每次观察，使用紫外线过滤器检测被钙氟污染的细胞壁和FITC过滤器来检测免疫化。为了更好地可视化结果，请使用 ImageJ 或类似的图像分析程序将每个紫外线滤镜图像与相应的 FITC 滤镜图像合并。通过精确定位特定表位的本地化，该协议能够对细胞壁组成进行定性。例如，1，5-阿拉比南在正在发育的Quercus子醚的细胞壁中是丰富的。很少消毒的同源体通常发现在Quercus子胚胎的根端，指定器官的机械性质。木龙存在于退化细胞中，例如在奎尔库斯亚橡子成熟期的最后阶段的内窥板细胞。JIM13 或 JIM8 识别的 AGP 表观存在于与生殖相关的结构上，例如与阿拉比多普西斯塔利亚纳的微游戏生成相关的细胞系，以及基底血管三叶草潜水器的耻辱性瘤和微生物。实施此协议时常见的错误通常很容易检测和识别。当洗涤被跳过或反应井被允许干燥时，次要抗体通常会以涂片出现。如果未绑定的原发抗体未正确冲走，将形成绿色荧色聚合物。折叠或分离的部分通常与由于使用不干净的滑梯或侵略性洗涤不良粘附有关。样品准备也至关重要。树脂块应没有裂缝，有清晰可见的淡黄色到浅棕色样品。低效嵌入式样品将显示粉状白点或区域。将样品尺寸保持在 8 毫米以下对于固定解决方案的渗透至关重要。固定不良的样品会出现深棕色或几乎黑色。温度过高会导致树脂破裂，使样品的分割变得不可能。免疫化技术的使用开辟了几个研究方向。人们可以继续使用更具体的技术，如细胞壁的生化分析，甚至进行分子方法。该技术提供的结果有助于理解细胞壁的组成，这种极其复杂的结构很难通过简单的化学分析来分析。

Research

JoVE Journal

Developmental Biology

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

荧光蛋白的表达<em&gt;披文昌鱼</em&gt;通过基因注射到未受精的卵母细胞

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

科研

发育生物学

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

新型乳腺脂肪移植技术可视化血管内皮干细胞的血管生成

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Live Imaging of Mouse Secondary Palate Fusion

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

拟南芥花器官和角果的全装清和染色

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

免疫荧光标记的微管和中心蛋白在体内肠道组织和 3D体外肠道 Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone

小鼠颅面组织和未切除骨骼的组织制备及免疫染色

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and ...

Use of Atomic Force Microscopy to Measure Mechanical Properties and Turgor Pressure of Plant Cells and Plant Tissues

利用原子力显微镜测量植物细胞和植物组织的机械特性和Turgor压力

We present here the use of atomic force microscopy to indent plant tissues and recover its mechanical properties. Using two different microscopes in ...

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

细胞细胞成像的微管细胞和微机械操作的 阿拉比多普西斯拍摄 阿皮克·梅里斯特姆

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in ...

Isolation of Endocardial and Coronary Endothelial Cells from the Ventricular Free Wall of the Rat Heart

从鼠心心室自由壁分离内膜细胞和冠状内皮细胞

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. ...