We are grateful to the technical support from Ms. Hong Xian Hsing, Mr. Min Jeng Li, and Mr. Eric C. P. Wu. We express our appreciation to Miranda Loney for English editing.

1.固定和嵌入<ol><li>试剂的准备<ol><li> 10倍磷酸盐缓冲盐水（PBS） <ol><li>添加80克NaCl，2克的KCl，14.4克Na 2 HPO 4，和2.4克KH 2 PO 4在800ml双蒸水（DDH 2 O）和将pH调节至7.4，用HCl。添加的DDH 2 O到1升的总体积，再加入1000微升焦碳酸二乙酯（DEPC），剧烈摇动。商店的PBS在室温下过夜并高压釜在121℃翌日20分钟。 </li></ol></li><li> 1X PBS <ol><li>稀释股票10X PBS中1:10的比例在DDH 2 O，得到的0.01M 的 Na 2的终浓度HPO 4，0.002M，KH 2 PO 4，0.003米氯化钾，和0.13 M氯化钠。 </li></ol></li><li>多聚甲醛（PFA）固定剂 注意:多聚甲醛是有毒的。在通风橱内准备。戴手套。 <ol><li>为了准备250毫升PFA固定剂，加热到100毫升1×PBS中，以70 - 80℃，并添加1750微升NaOH中。然后加入10多聚甲醛，并直至溶解调匀。放置在冰上的溶液中，然后将pH调节至7.2 的 H 2 SO 4（260 - 270微升为100毫升）中，冷却后。 </li><li>调节音量250ml的用1×PBS中，添加625微升戊二醛至0.25％的最终浓度。加入250微升的Triton X-100和250微升吐温20，以便于固定剂的渗入。 注:PFA固定剂可以储存于4℃一个月，而不会失去其定影能力。 </li></ol></li><li>制备不同浓度的乙醇（30％，50％，70％，85％，和95％）用DEPC处理过的水。 </li></ol></li><li>植物标本采集<ol><li>腋芽<ol><li>在四叶期使用成熟的蝴蝶兰植物。小心地撕开使用手中的中脉下面的叶子去掉叶子。 </li><li>用锋利的手术刀carefully从上单轴干的第三或第四叶的基部除去腋芽。 </li></ol></li><li>种子样品<ol><li>在授粉后4个月果实成熟兰豆荚。切豆荚纵向用手术刀。轻轻摇动开幕种子荚和释放在滤纸上的干种子。 </li></ol></li><li>球茎<ol><li>播种在1/2×Murashige和Skoog琼脂平板成熟兰花种子，并在一个12小时的光周期的组织培养室的温度和25℃的恒温生长。播种后7周品尝绿色球茎。 </li></ol></li><li>球茎状体（小巴） <ol><li>生长在作为相同的生长条件下，在步骤1.2.3.1先前10和地方描述T2再生琼脂平板兰花球茎。 8毫米 - 在5的高度收集10小巴。 </li></ol></li><li>叶样本<ol><li>收集来自成熟蝴蝶兰植物叶组织如在步骤中描述1.2.1.1。 </li><li>使用锋利的解剖刀从第二新开发叶切成小片的叶组织（7毫米长×5毫米宽）的。 </li></ol></li><li>根样本<ol><li>使用在步骤1.2.1.1中描述的相同的植物，解剖用锋利的解剖刀根尖组织为1厘米的长度。 </li></ol></li><li>幼穗<ol><li>用锋利的解剖刀切幼穗组织从花的前端部的长度秆10厘米。 </li></ol></li><li>花蕾<ol><li>消费税在5毫米直径从兰花梗的小花蕾。切花蕾组织纵向（3毫米厚）的一部分。 </li></ol></li><li>叶组织和谷类作物的组织小穗<ol><li>来自水稻，小麦和玉米植物的第一新开发的叶子切成1cm长叶片组织。 </li><li>收集来自植物10小穗（水稻，小麦和玉米）开花前一天。 </li></ol></li></ol></li><li>固定术<ol><li>通过将它们转移到含有15毫升的冰冷的PFA固定剂的玻璃闪烁小瓶立即修复植物样品。 </li><li>在4℃的室内阴凉处用真空（〜720毫米汞柱），植物样品。保持15真空 - 20分钟（小气泡应该从样品中被释放）。重复此步骤，直到大部分组织的真空释放后沉没。过夜保持真空和缓慢释放真空的第二天。 </li></ol></li><li>脱水 注:通过渗透步骤，使用从固定同一小瓶。倒或使用吸管流掉在先前步骤中的溶液，并用15毫升的新的解决方案到小瓶替换。取决于组织的纹理，治疗硬组织，例如兰花腋芽，种子，球茎，和PLB和水稻，小麦的小穗和玉米更长的时间;治疗较软的组织，例如兰花花苞，幼穗，叶和根，与水稻，小麦的叶和玉米的时间更短。 <o升&gt; <li>固定后，浸泡在15ml 1×PBS中的样品在冰上10分钟。 <ol><li>在室温下脱水的15ml乙醇一系列样品如下:30％乙醇30分钟，50％乙醇中30分钟，70％乙醇1小时，54分钟，提供更硬的组织和对于软组织30分钟85％的乙醇， 95％乙醇54分钟，提供更硬的组织和对于软组织30分钟，100％乙醇两次54分钟为硬组织和对于软组织30分钟。 注:植物性样品可以储存在70％的乙醇于4℃几个月。 </li></ol></li></ol></li><li>石蜡渗透<ol><li>渗透用15ml乙醇和二甲苯代替混合物样品每54分钟为硬组织和对于软组织30分钟，在室温下，如下所示:乙醇/二甲苯替代（2:1，V / V），乙醇/二甲苯替代（1 :1，v / v），乙醇/二甲苯代替（1:2，v / v），和纯二甲苯替代两次。注意:二甲苯是有毒的。不要在通风橱这一步。 </LI&gt; <li>在60℃下与在炉15毫升二甲苯替代和石蜡混合物小瓶渗入样品，对于硬组织过夜，并进行60分钟对于软组织如下:二甲苯代用品/石蜡（2:1，V / V），二甲苯代用品/石蜡（1:1，v / v），和二甲苯代用品/石蜡（1:2，v / v）。 </li><li>渗透用15毫升纯石蜡样品，一天两次，并在烘箱中在60℃下孵育。 </li><li>重复步骤1.5.3第二天。 </li></ol></li><li>组织包埋<ol><li>交换机上的组织包埋中心1小时的力量提前石蜡块包埋组织之前融化石蜡水库蜡。 </li><li>预热的变暖托盘的金属模（基3.3厘米长×2cm的宽度的尺寸）在62℃，并倒入约13毫升熔化的蜡的到模具基。 </li><li>从节1.5.4传送一个组织样品与温热镊子模具和定向它到所需的位置。 </li><li>移动模小心上凉板，并留下待蜡凝固。 </li></ol></li></ol> 2.组织切片<ol><li>石蜡切片法<ol><li>通过（在节1.6.2相同尺寸）放置一个包埋盒上的模具顶部使蜡的基础上，与熔融蜡填充和蜡固化后取下模具。 注:包埋盒将形成蜡基锚样品石蜡块。 </li><li>修剪石蜡块成适当的形状和大小，并把一些蜡件上的平铲。热火用酒精灯加热，直到蜡融化蜡块，然后将熔化的蜡在蜡基地2.1.1节坚持块。它夹住的切片。 </li><li>放置一个新的刀片到切片机，并调节角度到5度，以促进在切片机切片。 </li><li>切取试样的石蜡块切成薄片（10微米），如前面11所描述。 </li> &lt;/ OL&gt; </li><li>混合切割切法<ol><li>修剪从步骤1.6.4样品石蜡块在顶部带有一个梯形表面的柱子，以用刀片适当的大小。 </li><li>添加一些最佳切削温度化合物（OCT）的低温恒温舞台的中心。附加石蜡块到低温恒温器阶段，然后迅速定向组织块到所需的位置。 </li><li>转移蜡块/阶段低温恒温器室。华侨城允许在-42℃下速冻栏巩固10分钟。华侨城（ 图2C）的凝固过程中不要移动块。 </li><li>允许低温恒温器适配器和腔室温度至石蜡块/阶段附着到低温恒温器适配器之前冷却至C分别在-20℃和-16°。部组织至10微米厚。 </li><li>匹克组织切片用钳子。浮在800微升DEPC处理过的水部分在聚- L -赖氨酸COA泰德滑动并在42℃下将载玻片转移到热板上。 </li><li>允许各部分，以在42℃于DEPC处理过的水变平。使用滤纸从边缘把水沥掉。 </li><li>在幻灯片上的山组织通过将幻灯片上42℃的热板过夜。 </li></ol></li></ol> 3.组织染色<ol><li>脱蜡<ol><li>从热板收集的幻灯片，并将其放置在机架染色。添加150ml二甲苯中进通风柜一个染色缸和浸入二甲苯齿条5分钟。 </li></ol></li><li>补液<ol><li>在DEPC处理过的水制备不同浓度的乙醇溶液（100％，95％，70％，50％和30％）和补加入150ml溶液成不同的染色缸，分别。 </li><li>通过一系列递减的乙醇浓度，在室温下3分钟的每个步骤的再水合样品。在染色转移的幻灯片绞尽脑汁罐子另一种含罐子ðifferent乙醇浓度:100％乙醇，95％乙醇，70％乙醇，50％乙醇和30％乙醇。 </li><li>补液后，沉浸在DDH 2 O标本3分钟。 </li></ol></li><li>苏木素染色<ol><li>在苏木溶液中浸泡组织切片，1.5分钟，染色组织样本。 </li><li>简要地冲洗在DDH含有1 2 O - 2滴12N盐酸（HCl）的几秒钟，然后在DDH 2 O简要洗</li></ol></li><li>脱水<ol><li> 30％乙醇，50％乙醇，70％乙醇，95％乙醇和100％乙醇:通过一系列增加的乙醇浓度，在室温下3分钟的脱水样品。在通风柜5分钟二甲苯清除标本。 </li></ol></li><li>安装<ol><li>适当降600微升二甲苯类的安装介质上的幻灯片。小心地在样品盖玻片。避免气泡形成以获得良好品质的图像。 </li><li>允许的幻灯片空气干燥过夜。第二天观察在显微镜下试样。 注意:图像，在25X取决于试样的大小捕获到400X放大倍数。 </li></ol></li></ol> 4. 原位杂交<ol><li>探针合成<ol><li>克隆白蝴蝶兰肌动蛋白基因的特定使用的编码序列的引物5&#39;-GGCAGAGTATGATGAATCTGGTCC-3&#39;和5&#39;-AGGACAGAAGTTCGGCTGGC -3&#39;（获取242 bp的PCR扩增子）和CyclinB1的;使用1基因特异性编码序列的引物5&#39;-TCGTAGCAAGGTTGCTTGTG-3&#39;和5&#39;ATGAGCATGGCGCTAATACC-3&#39;（获取327 bp的PCR扩增子），为描述的12。 </li><li>根据制造商的协议结扎特定编码序列插入载体（ 例如，PGEMT）。 </li><li>产生地高辛（DIG）根据制造商的指示标记的使用SP6 / T7 DIG RNA标记试剂盒义和反义探针秒。 </li></ol></li><li> 原位杂交<ol><li>切2 和第 3腋芽组织切片（10微米厚）采用混合式切割方法生成，并安装到切片涂幻灯片。 </li><li>在二甲苯Deparaffinize组织切片（见3.1.1），补充水分递减的乙醇浓度（见3.2.2）中，在37℃用2毫克/毫升的蛋白酶K消化30分钟。 </li><li>根据协议来执行原位杂交先前描述11,13一些修改， 即 ，与杂交温度为肌动蛋白作为59℃和60℃ 中CyclinB1; 1杂交用40纳克DIG标记的RNA探针滑动。 </li><li>使用氮蓝四唑/ 5-溴-4-氯-3-吲哚基磷酸盐（NBT / BCIP）溶液如所述11以检测杂交信号。 </li></ol></li></ol>

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No conflicts of interest declared.

植物细胞有刚性细胞壁，坚韧的纤维，晶体，水分含量高，导致植物组织切片中组织撕裂的问题。即使基于石蜡的切片经常用于植物组织，内源晶体往往切片（ 图1）过程中撕裂植物组织。因为植物细胞内的水分含量固有的高的，基于低温恒温器，切片往往会造成细胞破壁和打击组织切片。 在本研究中，命名为混合式切割组合石蜡包埋和冷冻切片协议被开发并且获得良好质量的组织切片。该协议解决了通过引入石蜡包埋具有高水含量有关的问题，并减少了切片（ 图3）期间由低温下硬化石蜡的撕裂效应。因此，该修改的协议是在任一基于石蜡的sectio有利宁或cryosectioning用于保存植物组织的完整性。 这个手稿表明，混合式切割方法保留在蝴蝶兰的许多组织如腋芽，种子，和PLB 等含有高含量的结晶（ 图 3-4）的组织的完整性。此外，该协议是适合于谷物作物如生殖器官和水稻，玉米的叶，小麦含有高二氧化硅（ 图5-6）。据推测，该协议可以被应用到木质含高纤维植物。 一般来说，固定组织彻底是混合式切割非常重要的。我们发现，甲醛醇酸性酸（FAA）固定剂是比PFA保留一些顽抗组织的组织的完整性，如轴向芽，根， 等等。然而，PFA作品比联邦航空局在维护RNA的完整性更好。因此PFA建议修复样品原位杂交（ISH）的工作。这里所描述的协议被设计用于RNA ISH实验。因此，准备所有的试剂由DEPC处理消除了RNase污染，以避免RNA降解。如果混合形切割部分为解剖研究，定期反渗透（RO）水及其衍生缓冲器或试剂都是可接受的。 还原样品的大小和厚度小于3毫米为浸润有帮助的。此外，增加浸渍时间为脱水和渗透所必需的质地坚硬的组织。这个协议的限制可能由于由定影不足，脱水，和试样的渗透问题。因此，对于每个步骤的处理时间的调整是非常关键的，以产生质量好的石蜡块。通常情况下，很难组织需要更长的处理时间较软的组织。 此外，我们发现，混合式切割相结合的Wi成功合作日ISH提供选择的成绩单（ 图7）的空间分布。总之，这个协议是植物解剖学的研究有用并提供所选择的基因的组织特异性的RNA图谱。此外，它也可以应用于其他的分子研究如末端脱氧核苷酸转移的dUTP缺口末端标记（TUNEL）测定法， 荧光原位杂交（FISH），和免疫染色技术。总之，这种改进组织切片协议既是有益的，对植物群落的研究人员很有帮助。

的基于石蜡的切片方法，是解剖学研究一种广泛使用的技术。完整的组织解剖结构的保存是形态学和生物学研究的重要。石蜡包埋切片的技术是有利的，因为石蜡包埋保留的细胞和组织的形态。另外，石蜡块可以方便地用于长时间储存​​。然而，石蜡包埋切片不适合含有胞内晶体的植物组织。细胞内的晶体往往撕切片过程中石蜡彩带和损伤组织的完整性。 与石蜡切片，cryosectioning是比较快的，并部分可以不固定，串行脱水或包埋剂浸润1获得。冷冻切片与许多应用，例如免疫组织化学， 原位杂交和酶组织化学相容。 cryosectioning的另一优点是该方法不经过一个潜在的变性过程，如高温和化学处理，所以蜂窝分子保存完好的组织切片2内。 Cryosectioning一般优于石蜡切片基于对动物组织的研究。然而，cryosectioning并不在植物中的首选，因为冷冻温度有时会导致形成影响的部分完整性质量冰晶。尽管渗透调节，例如蔗糖溶液，聚乙二醇（PEG），或甘油3已报道降低冷冻条件下结晶冰的形成，其改进是不理想。 以适应不同的环境，不同的植物往往具有不同的组织质地和植物细胞进化以形成刚性细胞壁，坚韧纤维和结晶4,5-。例如，不溶性草酸钙晶体和二氧化硅机构是在相当普遍植物6。硅酸盐机构/晶体的报道，以帮助维持工厂结构，直立，防止病虫害的谷类植物7-9。 盆栽兰花切花兰花市场正在蓬勃发展，这是一个增长的行业。 白蝴蝶兰 （蝴蝶兰）是台湾最重要的出口观赏植物之一。显著已作出努力了解蝴蝶兰的兰花开花过程的形态和生理变化。 蝴蝶兰花的花尖峰从叶基部腋芽开始。一段凉爽环境温度后（约一个半月），腋芽放大，打破休眠，并且从叶基部突出培养成年轻花香尖峰。理解的生理，细胞和穗起始分子过程，重要的是开发一种健壮解剖技术visua丽泽组织或标记及时。然而，在兰组织普遍存在的晶体的存在，特别是在腋芽，使得解剖工作困难。 在这里，我们试图改善这种迄今已被视为技术上的挑战顽抗的植物组织部分的完整性。在这里我们命名为混合式切割的改进协议。它是使用低温恒温器进行的基于石蜡的切片方法。石蜡包埋解决植物组织中的含水量高。低温下切片变硬的石蜡块，减少了晶体撕裂的问题，并显著提高组织的完整性。该协议显著提高了顽抗的植物样品组织的完整性。

<table><tbody><tr><td>Embedding Center model EC780-1</td><td>CSA</td><td></td><td></td></tr><tr><td>Microtome model RM2255</td><td>Leica</td><td></td><td></td></tr><tr><td>Cryostat model CM 1950</td><td>Leica</td><td></td><td></td></tr><tr><td>Axio Scope A1 microscope&amp;nbsp;</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Murashige &amp;amp; Skoog medium including vitamins</td><td>Duchefa Biochemia</td><td>M0222.0050</td><td></td></tr><tr><td>RNase free surface decontaminant</td><td>Apex</td><td>10-228</td><td></td></tr><tr><td>Diethyl pyrocarbonate (DEPC)</td><td>Bio Basic Inc.&amp;nbsp;</td><td>D801154</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>MDBio, Inc.</td><td>101-7647-14-5</td><td></td></tr><tr><td>Potassium chloride, crystal (KCl)</td><td>J.T. Baker</td><td>7447-40-7</td><td></td></tr><tr><td>Sodium phosphate dibasic (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>7558-79-4</td><td></td></tr><tr><td>Potassium phosphate monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>7778-77-0</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich</td><td>16005</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Showa</td><td>19430160</td><td></td></tr><tr><td>Sulfuric acid (H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>7664-93-9</td><td></td></tr><tr><td>Glutaraldehyde solution</td><td>Sigma-Aldrich</td><td>111-30-8</td><td></td></tr><tr><td>Polyoxyethylene 20 sorbitan monolaurate (Tween 20)</td><td>J.T. Baker</td><td>9005-64-5</td><td></td></tr><tr><td>Octyl Phenol Ethoxylate (Triton X100)</td><td>J.T. Baker</td><td>9002-93-1</td><td></td></tr><tr><td>Glass scintillation vials</td><td>Newastar</td><td>DG60805-00020</td><td></td></tr><tr><td>Desiccator vacuum</td><td>Violet Bioscience Inc.</td><td>TS-402030</td><td></td></tr><tr><td>Rotary vane pump RZ2.5</td><td>Vacuubrand</td><td>36149406</td><td></td></tr><tr><td>Ethanol</td><td>J.T. Baker</td><td>64-17-5</td><td></td></tr><tr><td>Sub-X&amp;nbsp;</td><td>Leica Surgipath</td><td>3803670</td><td>Xylene substitute</td></tr><tr><td>Paraplast Plus&amp;nbsp;</td><td>Leica Surgipath</td><td>39602004</td><td>Tissue embedding paraffin</td></tr><tr><td>SUPERFROST micro slide glass</td><td>Matsunami</td><td>S7441</td><td></td></tr><tr><td>FSC 22 Clear&amp;nbsp;</td><td>Leica Surgipath</td><td>3801480</td><td>Optimal Cutting Temperature compound (OCT)&amp;nbsp;</td></tr><tr><td>Xylenes, Purified</td><td>Leica Surgipath</td><td>3803665</td><td></td></tr><tr><td>Hematoxylin</td><td>Merck</td><td>HX305311</td><td></td></tr><tr><td>Hydrochloric acid (HCl)</td><td>Sigma-Aldrich</td><td>7647-01-0</td><td></td></tr><tr><td>Micromount</td><td>Leica Surgipath</td><td>3801731</td><td>Mounting medium</td></tr><tr><td>pGEM T-easy vector</td><td>Promega</td><td>A1360</td><td></td></tr><tr><td>Proteinase K</td><td>Roche</td><td>3115879001</td><td></td></tr><tr><td>SP6/T7 DIG RNA Labeling Kit</td><td>Roche</td><td>11175025910</td><td></td></tr><tr><td>NBT/BCIP stock solution</td><td>Roche</td><td>11681451001</td><td></td></tr></tbody></table>

hybrid-cut: an improved sectioning method for recalcitrant plant tissue samples

维持植物部分完整性是用于在细胞，组织，或者甚至器官水平研究详细的解剖结构是至关重要的。然而，一些植物细胞有刚性细胞壁，坚韧的纤维和晶体 （草酸钙，硅石， 等 ），和水含量高，往往破坏植物组织切片中的组织的完整性。 该研究建立了一个简单的混合切割组织切片的方法。该协议修改一个基于石蜡的切片技术，提高了从不同的植物组织切片的完整性。植物组织在-16℃下在低温恒温器切片之前包埋在石蜡中。低温切片硬化石蜡块，减少撕裂和划伤，提高组织的完整性显著。该协议被成功地应用于富含钙草酸盐蝴蝶兰组织以及顽抗组织如生殖奥尔加纳秒和水稻，玉米和小麦的叶子。此外，从杂交切的组织切片的高品质，可以在组合原位杂交（ISH）用于提供感兴趣的基因的空间表达模式。总之，这个协议是用于含有高结晶或二氧化硅含量顽抗植物组织特别有用。质量好组织切片使形态学和其他生物的研究。

混合切提高组织切片的完整性 了解生殖花卉结构的解剖调查花芽分化的兰花的基本机制非常重要。然而，细胞内草酸钙晶体在蝴蝶兰的积累使得这些研究一项艰巨的任务。规避与在切片过程（ 图1）通过结晶撕裂引起相关联的问题，我们开发了一种我们命名为混合式切割系统。该协议结合了传统的石蜡包埋和冷冻切片技术。我们首先测试了混合削减蝴蝶兰腋芽，因为他们是臭名昭著的组织刚性和高晶体含量。腋芽组织固定在4％PFA（见协议，步1.3）。系列乙醇脱水和石蜡后，我nfiltration，腋芽包埋在石蜡块中。块切片（ 图2A）之前，修整成合适的尺寸。然后OCT的少量施加到低温恒温器阶段（ 图2B）的中心。然后将石蜡块粘附到通过OCT中的阶段。阶段是在一个低温恒温器保温10分钟，以允许OCT的完全凝固切片（ 图2C）之前，然后在-16进行冷冻切片 ℃的腔室（ 图2D）。 作为对比，含蝴蝶兰的腋芽石蜡块经常规切片机切片。如在图1A中所示，切片机切片后观察的石蜡色带的严重撕裂。的组织的完整性和细胞结构也受到影响（ 图1B）。混合剪切，另一方面，产生完整的组织切片（ 图3A）与保留结构完整性（ 图3B）。 应用混合切到P.各种组织阿芙罗狄蒂 为了进一步测试混合式切割的顺从，我们测试了蝴蝶兰的兰花不同的组织。它经常是很难获得，因为硬化种皮的具有良好的组织的完整性的种子部分。使用该协议，种子的详细结构切片（ 图4A）之后分别保存。如在图4A中 ，蛋白质体所示，公共存储产品14，可以清楚地识别。混合式切割也成功运行表明，一月龄球茎的芽顶端分生组织（从种子萌发的结构）（ 图4B）的详细结构和球茎样体（球茎， 图4C）。在PLB， 蝴蝶兰的兰花切片观察细胞内的晶体厚，肉质的叶子和他们执行景天酸代谢（CAM）型光合作用15。叶片的横向切片显示含木质部和韧皮部（ 图4D）大叶肉细胞和维管束。白天（ 图4D）中被观察到的叶背面上很少气孔的开口。事实上，CAM植物进化到最大限度的碳收益，但同时受干旱条件下15,16打开他们的气孔在夜间尽量减少水分流失。 P.根顶端分生组织阿芙罗狄蒂示于图4E。根尖细胞似乎含有结晶，切片（ 图4E）之后保存完好的显著数目。年轻的花卉尖峰纵向部分提供了信息离子对年轻的花香primordials（ 图4F）架构。此外，萼片，花瓣，唇瓣，和花粉可以清楚地从已经完成本5毫米花蕾（ 图4G）的分化年轻的花蕾纵截面确定。请注意，晶体的年轻花芽萼片积累。总之，这个协议的工作原理一致，以保持组织的完整性，并产生完整的形态使解剖和细胞可能的研究。 混合式切割用于保留杂粮组织的完整性 我们还测试杂交-切上谷类作物如水稻，小麦和玉米，包含高二氧化硅含量17,18。 如图5所示，水稻，小麦和玉米的叶子横向切片的组织的完整性被显著由海兰改进布里德切法。控制轧制的叶片，以避免水分流失的木质部，韧皮部，叶肉细胞，气孔，和泡状细胞清楚地水稻叶片部分鉴定。该克兰兹解剖，叶肉细胞和玉米叶片19,20的维管束被明确。这是有趣的发现气孔密度高两个玉米叶片（ 图5）的正面和背面的两侧。在玉米的0.7正面和背面气孔的比例已经先前21报道。此外，该协议还成功地工作，以提供从水稻，小麦和玉米（ 图6）小穗的详细的细胞形态。小穗已知含有丰富的二氧化硅9。通常情况下，将导致在导组织切片的困难。 原位杂交 在原位杂交（ISH）已经开发在组织水平11本地化的基因表达模式， 22-25。此外，原位杂交可以提供蜂窝，并且在一些情况下，亚细胞的mRNA分布在多细胞生物体26的分辨率。期间ISH，RNA和组织的完整性是必需，以获得关于所选择的转录可靠的空间信息。我们使用混合式切割协议测试ISH 肌动蛋白基因（PATC157348）使用引物5&#39;-GGCAGAGTATGATGAATCTGGTCC-3&#39;和5&#39;-AGGACAGAAGTTCGGCTGGC -3&#39;来获得242 bp的PCR扩增子细胞周期蛋白B1的克隆:1（PATC146999）基因特异性编码序列使用引物5&#39;-TCGTAGCAAGGTTGCTTGTG-3&#39;和5&#39;-ATGAGCATGGCGCTAATACC-3&#39;以获得327 bp的PCR扩增子。 ISH后， 肌动蛋白和Cyclin B1:1基因表达 在年轻而成熟腋芽监测 （ 图7）。这两个基因在2 次和第 3 次 腋芽分生组织细胞中表达，在第 3 次腋芽检测更强的信号。这些结果表明，混合式切割保留良好的解剖学和提供空间基因表达模式。 <img alt="图1" src="/files/ftp_upload/54754/54754fig1.jpg" /> 图1: 传统石蜡切片造成组织的严重撕裂含蝴蝶兰的腋芽石蜡块经常规切片机切片。传统的切片机切片后观察的石蜡色带的严重撕裂（A）的 </strong&gt;的信息。的组织的完整性和细胞结构也受到影响（B）。的箭头显示了组织切片的严重撕裂。箭头显示了晶体尸体。比例尺100微米。 <a href="http://ecsource.jove.com/files/ftp_upload/54754/54754fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54754/54754fig2.jpg" /> 图 2: 混合式切割切法的腋芽组织固定在PFA和组织进行脱水，用石蜡渗透，并嵌入石蜡块。石蜡块修整到合适的尺寸（A）。最佳切割温度化合物（OCT）是适用于低温恒温器级（B）的中心。石蜡块附着到OCT上低温恒温器阶段。在低温度下，蜡块<html>经华侨城（C）附着在低温恒温器阶段。组织切片在低温恒温室中于-16℃（D）的切片。比例尺代表0.5厘米（A），1厘米（BD）。 <a href="http://ecsource.jove.com/files/ftp_upload/54754/54754fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54754/54754fig3.jpg" /> 图3: 混合式切割提高诚信节含蝴蝶兰的腋芽石蜡块经混合式切割切片（A）和混合式切割法生产具有优良的组织的完整性（B）两部分。箭头表示嵌入在腋芽组织内源性结晶体。比例尺为100μm。rget ="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/54754/54754fig4.jpg" /> 图 4: 混合切到 蝴蝶 兰 的各种组织切片不同的兰花中的应用 组织中使用的混合式切割方法中，如在成熟阶段（A），球茎纵剖面（B）中 ，球茎状体的纵剖面（PLB）（C）中 ，横截面兰花种子的纵剖面剖开叶片（D），根（E）的纵切面，幼穗花（F），和年轻的花蕾（G）纵切面的纵向部分。组织切片用苏木精染色。 SC，种皮; PB，蛋白体;男，分生组织; MP，母亲PLB; DP，女儿PLB;广告，正面叶面上; AB，叶背面;圣，气孔; MC，叶肉细胞; VB，维管束; RC，根冠; FB，花芽; SE，萼片; PE，花瓣;香格里拉，唇瓣;宝，花粉;罗，顶突;钙，愈伤组织。箭头显示晶体。比例尺代表20微米（AE）和200微米（FG）。 <a href="http://ecsource.jove.com/files/ftp_upload/54754/54754fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54754/54754fig5.jpg" /> 图5:混合式切割，保留叶组织的完整性几种杂粮采用传统的石蜡法（左图），并在这项研究中（右图）开发的混合式切割技术叶组织的比较。图像显示水稻，小麦和玉米的叶横截面。 MC，叶肉细胞; pH值，韧皮部;圣，气孔; BC，BU<html> lliform细胞。箭头表示组织切片的撕裂。箭头表明硅体。蓝色虚线圆圈指示C4玉米克兰兹解剖。比例尺20微米。 <a href="http://ecsource.jove.com/files/ftp_upload/54754/54754fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54754/54754fig6.jpg" /> 图6: 混合式切割，保留小穗组织的完整性几种杂粮传统石蜡和混合式切割方法之间穗节组织的完整性比较。箭头表示组织切片的撕裂。箭头表明硅体。比例尺20微米（大米），200微米（小麦和玉米）。 <a href="http://ecsource.jove.com/files/ftp_upload/54754/54754fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> ENT"FO: - together.within页保留="1"&gt; <img alt="图7" src="/files/ftp_upload/54754/54754fig7.jpg" /> 图7: 肌动蛋白 和 CyclinB1的 原位杂交 ; 在 蝴蝶 兰的第二腋芽和第三腋芽的组织切片采用混合式切割方法制备的腋芽 1 表达模式 。共有40纳克肌动蛋白和CyclinB1蛋白的1 DIG标记探针用于杂交。感测探针被用作阴性对照。箭头指示腋芽分生繁殖。比例尺为100μm。 <a href="http://ecsource.jove.com/files/ftp_upload/54754/54754fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples at JoVE.com

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

混合式切割:用于顽拗植物组织样品的改进切法

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

hybrid-cut-an-improved-sectioning-method-for-recalcitrant-plant

Research

JoVE Journal

Biology

18.9K 次观看.  Academia Sinica. This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

视频: Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Thin Sectioning of Slice Preparations for Immunohistochemistry

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been preserved, freshly removed, or excised but kept viable, as in slice preparations of brain tissue.  Subsequent microscopic studies of this material can be challenging, as the tissue samples may be difficult to handle.  Demonstrated here is a method for obtaining thin cryostat sections of tissue with a thickness that may range from 0.2-5.0 mm.  We routinely cut 400 micron thick Vibratome brain slices serially into 5-10 micron coronal cryostat sections.  The slices are typically first used for electrophysiology experiments and then require microscopic analysis of the cytoarchitecture of the region from which the recordings were observed.  We have constructed a simple device that allows controlled and reproducible preparation and positioning of the tissue slice.  This device consists of a cylinder 5 cm in length with a diameter of 1.2 cm, which serves as a freezing stage for the slice.  A ring snugly slides over the cylinder providing walls around the slice allowing the tissue to be immersed in freezing compound (e.g., OCT).  This is then quickly frozen with crushed dry ice and the resulting  wafer  can be position easily for cryostat sectioning.  Thin sections can be thaw-mounted onto coated slides to allow further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry, as demonstrated here.

The present method allows reproducible cryostat sectioning of small, difficult-to-manage, tissue pieces, such as biopsies and brain slices. We utilize a simple aluminum freezing stage to facilitate handling of tissue and a standard cryostat to routinely produce 5-10 micron serial sections from 400 micron thick brain slices.

薄切片进行免疫组化切片的筹备工作

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been ...

科研

生物学

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundstr&#246;m at Jens.Sundstrom@vbsg.slu.se

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

非放射性原位挪威云杉杂交议定书适用的植物种类范围

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of ...

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a combination of light and electron microscopy. Samples embedded in resin are sectioned into arrays consisting of ribbons of hundreds of ultrathin sections and deposited on pieces of silicon wafer or conductively coated coverslips. Arrays are imaged at low resolution using a digital consumer like smartphone camera or light microscope (LM) for a rapid large area overview, or a wide field fluorescence microscope (fluorescence light microscopy (FLM)) after labeling with fluorophores. After post-staining with heavy metals, arrays are imaged in a scanning electron microscope (SEM). Selection of targets is possible from 3D reconstructions generated by FLM or from 3D reconstructions made from the SEM image stacks at intermediate resolution if no fluorescent markers are available. For ultrastructural analysis, selected targets are finally recorded in the SEM at high-resolution (a few nanometer image pixels). A ribbon-handling tool that can be retrofitted to any ultramicrotome is demonstrated. It helps with array production and substrate removal from the sectioning knife boat. A software platform that allows automated imaging of arrays in the SEM is discussed. Compared to other methods generating large volume EM data, such as serial block-face SEM (SBF-SEM) or focused ion beam SEM (FIB-SEM), this approach has two major advantages: (1) The resin-embedded sample is conserved, albeit in a sliced-up version. It can be stained in different ways and imaged with different resolutions. (2) As the sections can be post-stained, it is not necessary to use samples strongly block-stained with heavy metals to introduce contrast for SEM imaging or render the tissue blocks conductive. This makes the method applicable to a wide variety of materials and biological questions. Particularly prefixed materials e.g., from biopsy banks and pathology labs, can directly be embedded and reconstructed in 3D.

This protocol targets specific cells in tissue for imaging at nanoscale resolution using a scanning electron microscope (SEM). Large numbers of serial sections from resin-embedded biological material are first imaged in a light microscope to identify the target and then in a hierarchical manner in the SEM.

用于在大容量中查找特定细胞目标的串行剖面的多模式分层成像

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a ...

发育生物学

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

Here, we provide an easy, low-cost, and time-efficient protocol to chemically fix primate brain tissue with acrolein fixative, allowing for long-term preservation that is compatible with pre-embedding immunohistochemistry for transmission electron microscopy.

非人类灵长类动物脑组织的制备预嵌入免疫组化和电子显微镜

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and ...

神经科学

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

原位杂交技术在植物中的精确定位成绩单

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning

Sectioning of the paraffin-embedded tissue is widely used in histology and pathology. However, it is tedious. To improve this method, several commercial companies have devised complex section transfer systems using fluid water. To simplify this technology, we created a simple method using homemade equipment that combines cutting and floating within a simple thermostatic chamber; therefore, the sections automatically enter the water bath on the water surface. The hippocampus from adult mouse brains, adult mouse kidneys, embryonic mouse brains, and adult zebrafish eyes were cut using both conventional paraffin sectioning and the presented method for comparison. Statistical analysis shows that our improved method saved time and produced higher quality sections. In addition, paraffin sectioning of a whole specimen in a short time is easy for junior operators.

Here, we present a protocol to improve paraffin sectioning. This method combines cutting and floating using a simple thermostatic chamber to avoid the transfer process required by the conventional method. As a result, the efficiency and the number of intact paraffin sections were greatly improved.

切片中石蜡嵌入组织的切割和漂浮方法

Sectioning of the paraffin-embedded tissue is widely used in histology and pathology. However, it is tedious. To improve this method, several commercial ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

猪生物医学模型生物库组织样品的取样策略与处理

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

医学

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

激光捕获微分块RNA-测序空间和时间组织特异性基因表达分析

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

A Simple Dry Sectioning Method for Obtaining Whole-Seed-Sized Resin Section and Its Applications

The morphology, size and quantity of cells, starch granules&#160;and protein bodies in seed determine the weight and quality of seed. They are significantly different among different regions of seed. In order to view the morphologies of cells, starch granules and protein bodies clearly, and quantitatively analyze their morphology parameters accurately, the whole-seed-sized section is needed. Though the whole-seed-sized paraffin section can investigate the accumulation of storage materials in seeds, it is very difficult to quantitatively analyze the morphology parameters of cells and storage materials due to the low resolution of the thick section. The thin resin section has high resolution, but the routine resin sectioning method is not suitable to prepare the whole-seed-sized section of mature seeds with a large volume and high starch content. In this study, we present a simple dry sectioning method for preparing the whole-seed-sized resin section. The technique can prepare the cross and longitudinal whole-seed-sized sections of developing, mature, germinated, and cooked seeds embedded in LR White resin, even for large seeds with high starch content. The whole-seed-sized section can be stained with fluorescent brightener 28, iodine, and Coomassie brilliant blue R250 to specifically exhibit the morphology of cells, starch granules, and protein bodies clearly, respectively. The image obtained can also be analyzed quantitatively to show the morphology parameters of cells, starch granules, and protein bodies in different regions of seed.

This technique allows for the fast and simple preparation of whole-seed-sized resin section for the observation and analysis of cells, starch granules, and protein bodies in different regions of the seed.

获得全种子尺寸树脂部分的简单干截块方法及其应用

The morphology, size and quantity of cells, starch granules&#160;and protein bodies in seed determine the weight and quality of seed. They are ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

用于解释霉菌异养植物组织中真菌定植和种子共生萌发的显微镜技术

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...