组织化学染色<em>拟南芥</em>次生壁元素
Summary
Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.
Abstract
Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40–50%), hemicellulose (25–30%), and lignin (20–30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas Mäule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.
Introduction
植物细胞壁持有大量的信息及其各种部件之内：木质素，纤维素，半纤维素（木聚糖，葡糖醛酸，木葡聚糖，阿拉伯木聚糖，混合联动葡聚糖，或葡甘露聚糖），以及果胶。组织学技术提供学习辅助小区围墙内的差异在组织和细胞水平的重要的视觉线索。不同组织学技术被开发，可在文献中找到，但这些技术可以是具有挑战性和费时的初学者，因为用简单的视觉指示非常详细的协议很少，如果以往任何时候都可用。本研究的目的是为组织学染色技术简单的指引，以获得高质量的图像。
切片的茎组织中的细胞壁和细胞形状可视化的第一步。虽然手工切割的部分是价格便宜，需要较少的准备时间，使用vibratome的提供一致性和得到高品质的图像。使用vibratome允许产生更好的数据质量通过生成具有相同的厚度，这有助于产生清晰的图像和显著降低了生产样品之间不准确的差异，将通过一个坏的样品制备可简单地造成的危险，即使部分。使用树脂来修复新鲜标本可以对初学者是一个挑战，可能仍然是费时即使是专家的分析需要快速完成时。此外，它变得不可能测量任何生物活性与样品时，它已被埋入树脂。一个简单的技术，它采用琼脂糖和一个自制的模具是用于嵌入茎组织中有用，并且它也可以在需要的软组织解剖其它应用程序使用。相比，在树脂包埋的标本，该方法具有保持组织存活和减少样品操作的优点。通过vibratome切片组织是高度精确的，并生成均质项部分，其中，根据不同的研究的目标，然后可以用几种不同的染色技术中使用。
可视化的木质素和其它芳族化合物的最简单的方法，采用紫外线（UV）光。基于芳族化合物的分子被紫外光激发是一个古老的技术，但它仍然是最快的方法对木质素的可视化1。然而，紫外线的可视化其实是不理想的木质素检测，因为紫外线会激发其他芳烃。木质素主要由三个组成部分中，monolignols（hydroxycinnamyl醇：松柏醇，芥子醇，和对 -香豆醇）达1-3。间苯三酚染色可以提供线索，存在于木质部，纤维和气管组织4肉桂醛的程度。间苯三酚是一般的肉桂醛的一个良好指标，肉桂醛和芳香等可以区分。可以被检测到的芥子醇单体并通过区分使用马乌莱污点。甲苯胺蓝O是一个多色染料，因此具有染色细胞壁的不同元素以不同的颜色5,6的能力。主要使用的甲苯胺蓝O是检测果胶和木质素5,6。使用甲苯胺蓝澳的优点在于，细胞壁的许多元件可以在一个步骤中被可视化。这两种钙荧光白，刚果红是易于使用，可用于可视化纤维素。钙荧光白渍纤维素，胼胝质，和其他非取代的或弱取代的β-葡聚糖6-9，而刚果红染色直接向β-（1→4） -葡聚糖，特别是在纤维素10,11。这项研究的目标是提供简单的指引，使用上述染色技术，以从A获得高品质的图像拟南芥的茎。
Protocol
Representative Results
Discussion
拟南芥茎段被广泛用于研究细胞在次生壁的组织和定性研究野生型和转基因植物之间的差异。常用的技巧切片标本是直接手工切割;或当试样被嵌入在琼脂糖或固定剂，切片，可以用vibratome或切片进行。而相比之下，手工切割，最后两个允许减少生产样品之间的差异不准确的，将通过所引起的样品和不平坦的表面之间的厚度差的不良样品的制备可以简单地造成的风险。所有这些方法都有各?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢萨宾罗素编辑协助。 （http://www.jbei.org）支持由美国能源部，科学，生物和环境研究办公室，办公室这项工作是美国能源部联合生物能源研究所的一部分通过合同DE-AC02-05CH11231劳伦斯伯克利之间国家实验室和美国能源部门。
Materials
| Agarose | EMD | MERC2125 | CAS Number: 9012-36-6 |
| Phloroglucinol | Sigma | P 3502 | 1,3,5-trihydroxybenzene [CAS Number: 108-73-6] |
| Hydrochloric Acid | EMD | HX0603-75 | CAS Number: 7647-01-0 |
| Ammonium hydroxide | EMD | AX1303-6 | CAS Number: 1336-21-6 |
| Toulidine Blue O | Sigma | T3260 | Blutene chloride, Tolonium Chloride [CAS Number 92-31-9] |
| Potassium permanganate | Sigma | 223468 | CAS Number 7722-64-7 |
| Ethanol 190 proof | KOPTEC | V1401 | CAS Number: 64-17-5 |
| Congo Red | Sigma | C6277 | Disodium 3,3'-[[1,1'-biphenyl]-4,4'-diylbis(azo)]bis(4-aminonaphthalene 1-sulphonate) [CAS Number 573-58-0 ] |
| Fluorescent Brightener 28/ Calcofluor White Stain | Sigma | F3543 | 4,4'-Bis[4-[bis(2-hydroxyethyl)amino]-6-anilino-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonic acid [CAS Number 4404-43-7] |
| Vibratome | Leica | Leica Vibrating blade microtome VT1000S | http://www.leicabiosystems.com/products/sectioning/vibrating-blade-microtomes/details/product/leica-vt1000-s/ |
| Razor | American Safety razor company | Item # 60-0139-0000 | Stainless Steel Double Edge Blade (Personna Super) |
| Screw Cap Microcentrifuge Tubes (2ml) | VWR | 16466-044 | |
| Microcentrifuge Tubes (0.6ml) | Axygen Scientific | MCT-060-C | |
| Mitt | Bel-Art | 380000000 | SCIENCEWARE Hot Hand Protector Mitt |
| Tissue adhesive | Ted Pella Inc | 10033 | Store at 4°C or 20°C for 3 months or longer storage |
| Microwave | Panasonic | NN-SD762S | PELCO Pro CA 44 Instant tissue adhesive |
| Camera with CCD chip with no mechanical shutter | Hamamatsu | C4742-95 | |
| High speed color camera | QImaging | MicroPublisher 5.0 RTV | |
| Camera software | Molecular Devices | MetaMorph version 7.7.0.0 | |
| Imagining anaylsis | Adobe | Photoshop CS4 | |
| Micro Cover Glasses, Square, No.1 | VWR | 48366-067 | 22 x 22 mm (7/8 x 7/8")-Cover glasses are corrosion-resistant and uniformly thick and flat. No. 1 thickness is 0.13 to 0.17mm. |
| Frosted Micro Slides, 1mm | VWR | 48312-003 | 75 x 25 mm- 1mm |
| TX2 Filter cube | Leica | 11513851/11513885 | Filter used for Congo red analysis with a band-pass of 560/40. |
| Parafilm M | Alcan packaging | BRNDPM998 |
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