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。

1，干嵌入<ol><li>使在水中的7％的琼脂糖溶液（7克电泳级琼脂糖在100毫升蒸馏水中）。通过高压灭菌20分钟，或以最低的强度（ 例如 ，10％1250瓦特微波的强度）微波20分钟溶解琼脂糖。 </li><li>准备一个自制的模具嵌入茎用塑料瓶。 <ol><li>使用刀片，切断2毫升的螺旋盖离心管（A部分）的锥形底部。用注射器针头，穿刺在管帽上的孔比茎要被嵌入的直径稍大。接着，切0.5厘米了0.6 ml离心管（B部分）的底部。 </li><li>加0.6 ml离心管的截止0.5厘米部分送入预先切割2毫升微量离心管中。密封用封口膜（ 图1）的两个部分。注：该管的底部被切割，以便除去嵌入的样品后琼脂糖凝固。 Ŧ他孔盖将有助于保持稳定的干时干是嵌入在琼脂糖。该模具将有助于保持琼脂糖和嵌入时抱茎笔直。该封口膜将举行两部分，A和B，直到琼脂糖设置和茎被嵌入。 </li><li>用剃刀刀片，从感兴趣的区域（第一杆节间的例如中部）切茎段。注意：理想情况下，阀杆应只是嵌入，以避免破坏脱水前切断。可替换地，杆可以被暂时存储在含有水润湿滤纸的板。该板可以被放置在冰上直至准备将要被嵌入以防止组织脱水和变形。储存干时间超过30分钟没有长高湿度会导致干到干。 </li></ol></li><li>熔化琼脂糖在微波中在低强度。注意：含琼脂糖的瓶子可能会很热。使用手套拿起瓶子。让熔化的琼脂糖st和在室温下（在20°C和25°C），直到它冷却到约50℃。 </li><li>慢慢吸取5毫升琼脂糖入预制的塑料模具，填补了小瓶。注意：请确保没有气泡的模具。气泡会导致间隙中的琼脂糖模具，并不会完全覆盖干样品，这将最终导致试样的部分的切削不均匀。 <ol><li>让琼脂糖冷却30至60秒，成为半固体。用小牙签测试，确保它可以穿透并站起来，但不漂浮，在琼脂糖。注意：如果发现阀杆已放置在琼脂糖仍是热的会损坏样品通过干瘪的干，破坏细胞形态。 </li><li>将阀杆在此琼脂糖填充的小瓶。确保阀杆撑直。将多孔罩，所述阀杆的前端由盖保持这种方式。不要把螺丝帽。有时多于一个茎（单一种类的）可以是EMBEdded在一个小瓶;但在特定情况下，请不要使用上限。注意：如果螺丝帽被打开，阀杆会得到扭曲。 </li></ol></li><li>离开小瓶在室温或4℃下进行10-30分钟，直到它固化。 </li><li>通过轻轻地向外滑动管子取出模具。打开封口膜。用拇指按压模具的B部分的底部轻轻地从A部分释放凝固的琼脂糖到玻璃显微镜幻灯片。 </li><li>切琼脂糖嵌入阀杆分成三个大致相等的片为1.2厘米的长度。切出，这不是在琼脂糖和丢弃它的杆的一部分。 <ol><li>存储这些片断的琼脂糖与嵌入式茎在密闭的暗箱，4℃，直到准备第2 ml离心管中。另外，存储管在4℃下最多一个星期。注：存储是可能的，但实验者需要是嵌入式茎还活着，并根据实验需要注意，文物可能被引入。 </li></ol></li></ol> 2，干切片<ol><li>注意：请阅读vibratome手工精心并遵守所有安全指示。 </li><li>确保样品光盘是干净和完全干燥（无任何固体或液体）。清洁样品盘用刀片从实验前除去任何残余的胶和用水洗涤。再用干纸巾。注意：这将确保粘接效果很好，并结合到琼脂糖标本。如果这个步骤没有正确完成，它是可能的试样不附着到光盘上，并会导致以后的试样切片过程中脱落的圆盘。 <ol><li>用软纸擦拭，以从包含标本琼脂糖块中删除任何水分。 </li><li>放置一小滴组织粘合剂在试样​​圆盘。条纹列的粘接使用粘接瓶的前端覆盖在板的中间区域。很快到位琼脂糖块，以使标本要么垂直于或与板横向或纵向截面，分别平行。允许粘合剂试样固定在试样圆盘在室温或4℃下进行10-30分钟。注意：这是一个对时间敏感的一步。 </li></ol></li><li>修复到位样品盘上的缓冲盘或波谷。琼脂糖的与部分的块/ s应该是在与刀片平行。填充缓冲区托盘中，在室温蒸馏水，直到样品被完全淹没。 </li><li>切成两半的剃刀刀片，然后修剪的端部用坚固的剪刀，使刀片保持完全平坦的。附加一个半预切割刀片的上刀座。 <ol><li>上刀架设置角度为84°时，速度0.90毫米/秒（在vibratome位置8）和频率为50Hz（在vibratome位置5）。切成100微米厚的部分。使用连续模式。 NOTE：该厚度被选择，以确保该组织能够承受在一些染色方法中使用的各种酸和碱处理。对于切片设置窗口，并允许在连续模式下15-20分钟的vibratome节大约1.2厘米琼脂糖块。 </li></ol></li><li>切片在缓冲液盘过程中使用一次性塑料吸管收集部分。备选地，部分可以从缓冲托盘被转移到一个玻璃烧杯中，然后向一个明确的Petri板上。转几节到2.0 ml离心管中。注意：该标本收集从一个培养皿，因为它是更容易看清一个明确的背景的区域里，因为缓冲区托盘可引为下一个样品。 </li><li>的部分可被存储在一个50毫升管或收集和储存在1.5至2ml微量离心管中，在4℃下进行30至60分钟。注意：存放部分的时间较长，将导致图像质量变差。 </li&gt; </ol> 3，显微镜和照相机设置成像<ol><li>调整显微镜上的科勒照明。选择一个目标。专注于试样第一。带来的光强度的一半或更低。关闭视场光阑（FD）和孔径（AP）或把它带到在那个特定的目标尽可能低的设置。 <ol><li>在放大倍率较高，规模较大的环会。使用聚光调焦旋钮专注领域光圈一针见血越好。 </li><li>慢慢用冷凝器定心旋钮（围绕冷凝器针形螺钉）将视场光圈（六角形小环）的图像的中心。慢慢增加膜片（FD），使得该字段虹膜到达边缘。停止增加FD只是后场虹膜已通过边缘。 </li><li>慢慢地加大光圈（AP）来调整对比度。根据需要调节光的强度。注：科勒照明需要调整为每Õbjective，每一个实验的一天。注意调整为天之后所获得的数字。这些数字将目标得以来回切换的那一天，每次重复使用。 </li></ol></li><li>调整光圈，强度，曝光时间，增益和如表1中所述的过滤器，该信息应仅作为一个指南。 </li><li>使用高解析度数码相机采用了大尺寸隔行扫描CCD芯片，具有无机械快门来捕捉荧光图像;和一个高解析度，高速摄像机为亮场图像。 </li><li>过程中使用标准软件的幻灯片。装载软件。点击“获取”和“设置摄像头/板”，然后选择要使用的相机。单击“确定”。点击“获取”，其次是“颜色标签”。在“颜色标签”，选择“拜耳法”，其次是“平均四”。添加建议的增益（红，绿，蓝）来回米表1。选择“保存设置”选项卡，并单击其中的图像将被存储的目录。从表1中的建议设置“曝光时间”设置“分级”在“1”和“实时分级”为“1”。专注于感兴趣的区域，然后点击“保存活的。” </li><li>在相机软件输出的图像是在“TIF”格式。分析的细胞类型的组织化学中的图像。使用标准的计算机软件演讲和出版物的下游用途。 </li></ol> 4，紫外线和明场成像<ol><li>用1毫升移液管的移液管尖切，使得部分可以很容易地吸出。轻轻移液管部分到尖端和上至显微镜载玻片。盖上盖玻片的部分。观察在紫外光下或明场照明部分。 </li></ol> 5，间苯三酚 -盐酸（威斯纳）染色12 <ol><li>溶解0.3克间苯三酚在10毫升无水乙醇中，制成3％的间苯三酚溶液。混合浓盐酸（37 N）的一个容积为两卷乙醇3％间苯三酚的;这个解决方案是间苯三酚 - 盐酸（PH-HCl中）或威斯纳污点。注意：该解决方案是要作出关于染色的天新鲜和不能存储，因为该解决方案会随着时间的推移和染色将变得无效。注意：盐酸具有很强的腐蚀性，所以使用非常谨慎，而处理PH-盐酸污点。 </li><li>转移茎段至2.0ml微量离心管中。加入1 ml的PH-HCl溶液中含有的部分的管子，立即盖住它，因为HCl的pH值 - 盐酸污点是高度腐蚀性的。轻轻移动管，以确保所有的部分都染。对此的一种替换步骤是保持管帽打开，使得PH-HCl溶液中，可以用移液管上下，优选不干扰秒系统蒸发散。注意：要小心，不要将部分吸取到枪头，因为这可能会损坏这些部分。 <ol><li>用1毫升移液管的移液管尖切以这样一种方式，该部分可以很容易地吸出无损坏。轻轻吸取部分插入尖端和在显微镜载玻片上。盖上盖玻片的部分。根据观察明场照明部分。注：将pH-HCl溶液干涸在5-10分钟，使试样的劣化。因此，成像，必须在这段时间内完成。 </li></ol></li></ol> 6，马乌莱染色13,14 <ol><li>溶解的0.2g高锰酸钾在40毫升蒸馏水中，制成0.5％的高锰酸钾溶液。储存在黑暗的瓶子在室温下最多7天。 <ol><li>加入1 ml浓HCl（37 N）至9毫升蒸馏水水（1:10）准备一个3.7％的HCl溶液。准备在一个新鲜的3.7％HCl溶液天实验。确保所使用的37 N的HCl原液是不是太旧了。浓氢氧化铵溶液（14.8 M）应保存在4℃，以防止饱和溶液的摩尔浓度的降低。 </li></ol></li><li>转移茎段至2.0ml微量离心管中。加入1毫升0.5％的高锰酸钾溶液至含有部分管子。 <ol><li>移液器的0.5％高锰酸钾溶液，上下轻轻的，最好不干扰部分。孵育2分钟，并重复移液。让微量离心管支架，直到所有的部分安顿下来。注意：要小心，不要将部分吸取到枪头的部分可能被损坏。这可能是很难看到的茎段作为解决方案是黑暗的，所以保持管与试管架的部分，并允许他们定居2分钟。 </li><li>用1毫升移液管，抽出700微升0.5％的高锰酸钾溶液。加入700μl蒸馏水冲洗出来的高锰酸钾溶液。重复3至4倍，或直至水溶液保持清晰。 </li><li>倒掉水。快速加入1 ml 3％的HCl，直到深褐色的颜色是从部分放电。这可能在3-5分钟发生或可能需要洗涤2次的每次5分钟。吸取了所有的3％HCl溶液，并立即进行下一个步骤。 </li><li>加入1毫升浓氢氧化铵溶液。用1毫升移液管的尖端切割以这样一种方式，该部分可以很容易地吸出无损坏。轻轻吸取部分插入尖端和在显微镜载玻片上。盖上盖玻片的部分。根据观察明场照明部分。注意：由于氢氧化铵是腐蚀性极强，它需要额外的关怀和照顾，以避免造成腐蚀显微镜或镜头的阶段。注：氢氧化铵烟雾会起雾盖玻片，因此很难观察到样品。因此要格外小心加入盖玻片之前。该溶液干涸在5-10分钟，使摄像，必须在这段时间内完成。 </li></ol></li></ol> 7，刚果红染色11,15 <ol><li>溶解0.5克刚果红在100毫升蒸馏水中，制成0.5％的刚果红溶液。 </li><li>转移茎段至2.0ml微量离心管中。加入1毫升0.5％的刚果红溶液的管构成。轻轻吸取的0.5％刚果红溶液向上和向下，而不会干扰部分。注意：小心不要吸取了部分成枪头，因为这可能会损坏的部分。 <ol><li>在室温下孵育10分钟，并重复移液。跟随孵育在室温下另外3分钟。 </li><li>用1毫升吸管绘制出700微升0.5％刚果红溶液。加入700微升的蒸馏水冲洗出来的刚果红溶液。重复洗涤3至4倍，直到洗涤液是明确的。 </li><li>用1毫升移液管，其前端切以这样一种方式，该部分可以很容易地吸出无损坏。轻轻吸取部分插入尖端和在显微镜载玻片上，用盖玻片覆盖。使用过滤器与560/40带通观察样品下蓝色光激发显微镜幻灯片。注意：不要将水的部分为长之前，微观分析，因为水会洗出的刚果红在10-30分钟。 </li></ol></li></ol> 8，钙荧光白染色9 <ol><li>在蒸馏水中制备出0.2％原液10毫升溶解0.02克荧光增白剂28（钙荧光白M2R）。的0.2％的溶液可在4℃下存放在一个黑暗的瓶子6个月通过稀释0.2％的储备溶液的10倍，用蒸馏水制备的0.02％的工作溶液。 </li><li>转移茎段至2.0ml微量离心管中。添加到管1毫升0.02％的calcofluor白色的解决方案。轻轻吸取了0.02％钙荧光白液上下。注意：小心不要吸取了部分成枪头，因为这可能会损坏的部分。 <ol><li>在室温下孵育5分钟，并重复移液。孵育部另外3分钟，在室温下进行。注：允许部分沉降到管的底部，以使其更容易吸取出来的溶液，而不会损坏部分。 </li><li>用1毫升吸管绘制出700微升0.02％钙荧光白液，并加入700微升的蒸馏水;等待5分钟，冲洗出来的钙荧光白的解决方案。重复洗涤3至4倍。用1毫升吸管用枪头切如方式，部分可以很容易地吸出无损坏。 </li><li>轻轻吸取部分插入尖端和在显微镜载玻片上并用盖玻片覆盖。观察在紫外灯下的部分。 </li></ol></li></ol> 9，甲苯胺蓝E O染色16,17 <ol><li>溶解0.02克甲苯胺蓝O输入100毫升蒸馏水中，制备0.02％溶液。注：甲苯胺蓝O解决方案可以存储两周在黑暗的瓶子在室温下。 </li><li>转移茎段至2.0ml微量离心管中。加入1毫升0.02％的甲苯胺蓝O解决方案到管。轻轻吸取了0.02％甲苯胺蓝O解决方案上下。然后在室温下孵育5分钟，并重复移液一次。注意：小心不要吸取了部分成枪头，因为这可能会损坏的部分。让微量离心管站在原地，直到部分安顿下来。这可能是很难看到，因为该解决方案是黑暗的，所以保持管中的试管架的部分，并允许他们定居2分钟在室温下。 <ol><li>用1毫升吸管绘制出700微升0.02％甲苯胺蓝O解决方案。加入700μl蒸馏水冲洗出来的甲苯胺蓝Ø溶胶ution。重复3至4倍，或直至洗涤液是明确的。用1毫升移液管的移液管尖切以这样一种方式，该部分可以很容易地吸出而不损坏它们。 </li><li>轻轻吸取部分插入尖端和在显微镜载玻片上并用盖玻片覆盖。根据观察明场照明部分。 </li></ol></li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+in+the+interaction+of+direct+dyes+with+polysaccharides.">Specificity in the interaction of direct dyes with polysaccharides.</a> Carbohydrate Research. 85, 271-287 (1980).</li><li>Hughes, J., McCully, M. 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+use+of+an+optical+brightener+in+the+study+of+plant+structure.">The use of an optical brightener in the study of plant structure.</a> Stain technology. 50, 319-329 (1975).</li><li>Verbelen, J. P., Kerstens, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarization+confocal+microscopy+and+congo+red+fluorescence:+a+simple+and+rapid+method+to+determine+the+mean+cellulose+fibril+orientation+in+plants.">Polarization confocal microscopy and congo red fluorescence: a simple and rapid method to determine the mean cellulose fibril orientation in plants.</a> Journal of microscopy. 198, 101-107 (2000).</li><li>Anderson, C. T., Carroll, A., Akhmetova, L., Somerville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+cellulose+reorientation+during+cell+wall+expansion+in+Arabidopsis+roots.">Real-time imaging of cellulose reorientation during cell wall expansion in Arabidopsis roots.</a> Plant physiology. 152, 787-796 (2010).</li><li>Liljegren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phloroglucinol+Stain+for+Lignin.">Phloroglucinol Stain for Lignin.</a> Cold Spring Harbor Protocols. , (2010).</li><li>Nakano, J., Meshitsuka, G., lin, S., Dence, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Detection of Lignin Methods in Lignin Chemistry. , (1992).</li><li>Sibout, R., 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=CINNAMYL+ALCOHOL+DEHYDROGENASE-C+and+-D+are+the+primary+genes+involved+in+lignin+biosynthesis+in+the+floral+stem+of+Arabidopsis.">CINNAMYL ALCOHOL DEHYDROGENASE-C and -D are the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis.</a> The Plant cell. 17, 2059-2076 (2005).</li><li>Teather, R. M., Wood, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Congo+red-polysaccharide+interactions+in+enumeration+and+characterization+of+cellulolytic+bacteria+from+the+bovine+rumen.">Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen.</a> Applied and environmental microbiology. 43, 777-780 (1982).</li><li>Yeung, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+beginner's+guide+to+the+study+of+plant+structure.">A beginner's guide to the study of plant structure.</a> Proceedings of the 19th Workshop/Conference of the Association for Biology Laboratory Education (ABLE. 19, 365-36 (1998).</li><li>Turner, S. R., Somerville, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collapsed+xylem+phenotype+of+Arabidopsis+identifies+mutants+deficient+in+cellulose+deposition+in+the+secondary+cell+wall).">Collapsed xylem phenotype of Arabidopsis identifies mutants deficient in cellulose deposition in the secondary cell wall).</a> The Plant Cell Online. 9, 689-701 (1997).</li><li>Iiyama, K., Pant, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+the+M&#228;ule+colour+reaction.+Introduction+of+methylated+syringyl+nuclei+into+softwood+lignin.">The mechanism of the M&#228;ule colour reaction. Introduction of methylated syringyl nuclei into softwood lignin.</a> Wood Sci. Technol. 22, 167-175 (1988).</li><li>Yang, F., 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=Engineering+secondary+cell+wall+deposition+in+plants.">Engineering secondary cell wall deposition in plants.</a> Plant biotechnology journal. 11, 325-335 (2013).</li><li>Zhong, R., Ripperger, A., Ye, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+deposition+of+lignin+in+the+pith+of+stems+of+two+Arabidopsis+mutants.">Ectopic deposition of lignin in the pith of stems of two Arabidopsis mutants.</a> Plant physiology. 123, 59-70 (2000).</li><li>Chapple, C. C., Vogt, T., Ellis, B. E., Somerville, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Arabidopsis+mutant+defective+in+the+general+phenylpropanoid+pathway.">An Arabidopsis mutant defective in the general phenylpropanoid pathway.</a> The Plant cell. 4, 1413-1424 (1992).</li><li>Fagard, 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=PROCUSTE1+Encodes+a+Cellulose+Synthase+Required+for+Normal+Cell+Elongation+Specifically+in+Roots+and+Dark-Grown+Hypocotyls+of+Arabidopsis.">PROCUSTE1 Encodes a Cellulose Synthase Required for Normal Cell Elongation Specifically in Roots and Dark-Grown Hypocotyls of Arabidopsis.</a> The Plant Cell Online. 12, 2409-2423 (2000).</li></ol>

The authors have no financial conflicts to disclose.

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?...

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。

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			<td>P 3502</td>
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			<td>T3260</td>
			<td>Blutene chloride, Tolonium Chloride [CAS Number 92-31-9]&#160;</td>
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			<td>Sigma</td>
			<td>223468</td>
			<td>CAS Number 7722-64-7&#160;</td>
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			<td>Ethanol 190 proof</td>
			<td>KOPTEC</td>
			<td>V1401</td>
			<td>CAS Number: 64-17-5</td>
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			<td>Congo Red</td>
			<td>Sigma&#160;</td>
			<td>C6277</td>
			<td>Disodium 3,3&#39;-[[1,1&#39;-biphenyl]-4,4&#39;-diylbis(azo)]bis(4-aminonaphthalene 1-sulphonate) [CAS Number 573-58-0 ]</td>
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			<td>Fluorescent Brightener 28/ Calcofluor White Stain</td>
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			<td>American Safety razor company</td>
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			<td>Stainless Steel Double Edge Blade (Personna Super)</td>
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			<td>Camera with CCD chip with no mechanical shutter&#160;</td>
			<td>Hamamatsu</td>
			<td>C4742-95</td>
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			<td>High speed color camera&#160;&#160;&#160;</td>
			<td>QImaging</td>
			<td>MicroPublisher 5.0 RTV</td>
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			<td>Camera software&#160;</td>
			<td>&#160;Molecular Devices</td>
			<td>MetaMorph version 7.7.0.0</td>
			<td></td>
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			<td>Imagining anaylsis&#160;</td>
			<td>Adobe&#160;</td>
			<td>Photoshop CS4</td>
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			<td>Micro Cover Glasses, Square, No.1</td>
			<td>VWR</td>
			<td>48366-067</td>
			<td>22 x 22 mm (7/8 x 7/8&#34;)-Cover glasses are corrosion-resistant and uniformly thick and flat. No. 1 thickness is 0.13 to 0.17mm.&#160;</td>
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			<td>Frosted Micro Slides, 1mm</td>
			<td>VWR</td>
			<td>48312-003</td>
			<td>75 x 25 mm- 1mm</td>
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			<td>TX2 Filter cube</td>
			<td>Leica</td>
			<td>11513851/11513885</td>
			<td>Filter used for Congo red analysis with a band-pass of 560/40.</td>
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			<td>Parafilm M</td>
			<td>Alcan packaging</td>
			<td>BRNDPM998</td>
			<td></td>
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</table>

histochemical staining of arabidopsis thaliana secondary cell wall elements

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&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;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 M&#228;ule 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.

干嵌入和切片： 使用自制的塑料模具中嵌入的杆在7％琼脂糖证明是快速和容易的（ 图1）。这两个部分（A和B; 图1）嵌入式小瓶系统变得简单轻松地释放嵌入琼脂糖的琼脂糖不会粘到小瓶部分是惰性干，保持系统清洁。小瓶可以多年重复使用多次。存储嵌入式干的便利也使得接下来的步骤更简单。为了节省某个时候，类似的嵌入式茎可以分组（最多3个）在一个切片?...

Watch this Scientific Journal Video about Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements at JoVE.com

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

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.

组织化学染色<em&gt;拟南芥</em&gt;次生壁元素

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

histochemical-staining-arabidopsis-thaliana-secondary-cell-wall

Research

JoVE Journal

Biology

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

40.2K 次观看.  Joint Bioenergy Institute. 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.

视频: Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

从14日龄的幼苗组织原生质体的分离拟南芥</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

科研

生物学

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&amp;#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

花香浸改造拟南芥检查 pTSO2：β-葡萄糖醛酸酶报告基因的表达

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

高效，快速的早期胚胎分离<em&gt;拟南芥</em&gt;种子

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

结晶纤维素积累高分辨率的量化<em&gt;拟南芥</em&gt;根监视特定组织细胞壁修改

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light conditions, which are preferably variable in quantity and quality so that studies under different setups can be conducted. These requirements are met by climatic chambers featuring light emitting diodes (LED) lights, which can &#8211; in contrast to fluorescent lights &#8211; be set to different wavelengths. LEDs are energy conserving and emit virtually no heat even at light intensities, which often constitutes a problem with other light sources. The presented protocol provides a step-by-step guidance of how to program a climatic chamber equipped with variable LED lights as well as describing several approaches for in depth analysis of growth phenotypes. Depending on the experimental set-up various characteristics of the growing plants can be observed and analyzed. Here we describe how to determine fresh weight, leaf area, photosynthetic activity, and stomatal density. We demonstrate that in order to obtain reliable data and draw valid conclusions it is mandatory to use a sufficient number of individuals for statistical evaluation. Taking too few plants for this kind of analysis results in high statistical errors and consequently in less clear interpretations of the data.

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Here, we present a protocol for studying plant growth behavior and especially phenotypes in a reproducible manner. We show how to provide variable and at the same time stable light conditions. Proper analyses depend on sufficient sample numbers and valid statistical evaluations.

不同光照条件下的拟南芥生长行为分析

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light ...

通过共聚焦成像对 ERL1 动力学进行时空研究

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

作者聚焦：研究气孔谱系细胞中膜运输事件的基于图像的方法  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

基于图像的方法研究气孔谱系细胞中的膜运输事件

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...

一种测量拟南芥花粉水合作用曲线的改进方法

A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is established between the interacting partners. Studies across a range of species have revealed that a series of molecular checkpoints regulate the pollen-stigma interaction to ensure that only compatible, generally intraspecific pollen is successful in effecting fertilization. In species that possess a 'dry stigma', such as the model plant Arabidopsis thaliana, the first post-pollination, prezygotic compatibility checkpoint is the establishment of pollen hydration. 
This phase of pollination is tightly regulated, whereby signals from the pollen grain elicit the release of water from the stigma, thus permitting pollen hydration. The ability to accurately measure and track pollen hydration over time is key to the design of experiments directed at understanding the regulation of this critical step in reproduction. Published protocols frequently utilize flowers that have been excised from the parent plant, maintained on liquid or solid media, and bulk pollinated. 
This paper describes a noninvasive, in vivo pollination bioassay that permits minute-by-minute hydration tracking of individual A. thaliana pollen grains at high resolution. The assay is highly reproducible, able to detect very subtle variations of pollen hydration profiles, and thus is suitable for the analysis of mutants that affect pathways regulating pollination. Although the protocol is lengthier than those described for bulk pollinations, the precision and reproducibility it provides, along with its in vivo nature, make it ideal for the detailed dissection of pollination phenotypes.

作者聚焦：拟南芥的高分辨率、单粒、体内花粉水合生物测定  

An improved method to measure pollen hydration profiles in Arabidopsis thaliana is described here. The new method offers higher resolution, is noninvasive, and is highly reproducible. The protocol represents a new tool for a finer dissection of the processes that regulate the early stages of pollination.

拟南芥的高分辨率、单粒、体内花粉水合生物测定

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is ...