Name: a protocol for immunohistochemistry and rna in-situ distribution within early drosophila embryo
Uploaded: 2022-05-06T15:00:00
Description: Watch this Scientific Journal Video about A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo at JoVE.com

这项工作得到了国家自然科学基金（#31771377/31571273/31371256），国家教育部外国杰出科学家项目（#MS2014SXSF038），国家教育部中央高校研究基金（#GK201301001/201701005/GERP-17-45）的支持，XZ由优秀博士论文基金（#2019TS082/2019TS079），陕西省教育厅重点项目（#20JS138） 支持。 陕西省科技厅自然科学基础研究计划青年项目（#2020JQ-885）。

1. 葡萄汁琼脂平板的制备
<ol><li>将5克琼脂和5克蔗糖加入150毫升蒸馏水中。用微波炉煮沸，直到琼脂和蔗糖完全溶解。将 50 mL 100% 葡萄汁和溶液混合在一起。</li>
	<li>加入1 mL 100%丙酸，使最终浓度为0.5%丙酸。将25mL准备好的溶液倒入每个板中。琼脂凝固后，将培养基储存在4°C。</li>
</ol>2. 涂布载玻片
<ol><li>用洗涤剂预处理载玻片。用自来水清洗载玻片3?...

<ol>
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RyRs和IP3Rs介导的钙信号传导是脊椎动物和无脊椎动物许多生理和病理过程的基本途径1，2，3，4。在人类中，RyR2基因中的点突变（例如CPVT相关的R4496C突变）导致心肌细胞肌质网的钙泄漏，从而导致心脏功能障碍。这些突变表现为心律失常，在体力活动期间具有心脏性猝死的高风险，并且在设计用于携?...

钙诱导钙释放信号传导（CICR）在许多生物过程中起着至关重要的作用，例如骨骼肌/平滑肌和心脏血管功能，细胞增殖和凋亡，发育，衰老，神经元突触可塑性和再生1，2，3，4，5，6.Ryanodine受体（RyRs）和肌醇1，4，5-三磷酸受体（IP3Rs）是由其调节蛋白激酶A（PKA），Ca2 +/钙调蛋白依赖性蛋白激酶II（CaMKII），FK506结合蛋白（FKBPs），钙马蛋白（CSQ），三星素和junctin1，2，3，4，5，6控制的钙信号通路中的主要参与者.这些蛋白质的异常人类表达和突变可导致病理生理学，如心律失常7和致癌增殖8，9。
FKBPs通过RyR调节内质网状（ER）的钙释放。该过程对于收缩机制至关重要，因此负责肌球蛋白收缩通过钙诱导的钙释放以及胚胎RyRs1，2产生的所有机械运动。在小鼠模型中，缺乏RyR2及其调节因子FKBP12 / Calstabin总是致命的，无论是在胚胎发育期间还是在产后早期10，11，12。FKBP12 / Calstabin敲除小鼠在胚胎发育过程中表现出严重的心脏缺陷，具有不规则的兴奋 - 收缩耦合（EC）和脑水肿。这表明FKBP12 / Calstabin在调节RyR2通道表达中起着至关重要的作用，这对心脏和大脑发育都很重要10。
RyR传导的钙火花最初是在受精的Medaka卵的受精卵形成阶段发现的13，14。然而，对钙信号传导在早期胚胎发育中的作用的研究很少。在 黑色果蝇中，从 DmFKBP12 S107突变体获得的结果提供了强有力的证据，支持该基因对幼虫发育和健康寿命的重要性，这归因于其对抗氧化应激的功能15，16。最近，我们确定了 FKBP12 / Calstabin 蛋白和信使RNA在早期 果蝇黑色素胃发育 过程中的动态定位17。使用该方法中描述的方法，我们能够在合胞体胚芽孢子虫（0-2小时），细胞胚芽肿（2-3小时），早期胃肠（3-12小时）和晚期胃肠炎（12-24小时）期间跟踪D . melanogaster 中FKBP12 / Calstabin的表达。在本文中，我们介绍了先前研究中每种方法的详细方案，包括用于经典石蜡切片的胚胎前包埋，用于胚胎切片的预包衣载玻片处理，组织化学染色和免疫染色以及用于鉴定基因表达的mRNA 原位 杂交。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>-20&#176;C Refrigerator</td>
			<td>Meiling Biology &#38;Medical</td>
			<td>DW-YL270</td>
			<td>Used for regent storage</td>
		</tr>
		<tr>
			<td>-80&#176;C Ultra low temperature refrigerator</td>
			<td>Thermo</td>
			<td>Forma 90 Series</td>
			<td>Used for regent storage</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>WXBB6360V</td>
			<td>Preparation of grape juice agar plates</td>
		</tr>
		<tr>
			<td>Anti-Digoxigenin-AP, Fab fragments</td>
			<td>Roche</td>
			<td>11093274910</td>
			<td>For the detection of digoxigenin-labeled compound</td>
		</tr>
		<tr>
			<td>Biochemical incubator</td>
			<td>Shanghai Bluepard Instruments</td>
			<td>LRH-250</td>
			<td>In-situ Hybridization</td>
		</tr>
		<tr>
			<td>Bouin&#39;s solution</td>
			<td>Sinopharm Chemical Reagent at Beijing</td>
			<td>69945460</td>
			<td>Drosophila Embryo Embedding</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>540BH07808</td>
			<td>In-situ Hybridization</td>
		</tr>
		<tr>
			<td>Centrifuge tube</td>
			<td>Denville</td>
			<td>C-2170</td>
			<td>Drosophila Embryo Collection</td>
		</tr>
		<tr>
			<td>Chrome Alum</td>
			<td>Sinopharm Chemical Reagent at Beijing</td>
			<td>10001018</td>
			<td>Coating Slides</td>
		</tr>
		<tr>
			<td>Constant temperature water bath</td>
			<td>Jintan Henfeng Instruments</td>
			<td>KW-1000DC</td>
			<td>Hematoxylin-Eosin Staining, Immunohistochemistry, In-situ Hybridization and Periodic Acid-Silver Methenamine Staining</td>
		</tr>
		<tr>
			<td>Dako REAL EnVision Detection System</td>
			<td>Dako</td>
			<td>K5007</td>
			<td>In immunohistochemical reaction or in situ hybridization reaction, it binds to the primary antigen antibody, and the target is labeled by staining.</td>
		</tr>
		<tr>
			<td>DEPC</td>
			<td>Sigma-Aldrich</td>
			<td>D5758</td>
			<td>In-situ Hybridization</td>
		</tr>
		<tr>
			<td>DIG RNA Labeling Kit</td>
			<td>Roche</td>
			<td>11093274910</td>
			<td>RNA labeling with diagoxigenin-UTP by in vitro transcription with SP6 and T7 RNA polymerase</td>
		</tr>
		<tr>
			<td>Drosophila melanogaster</td>
			<td>Bloomington Stock Center</td>
			<td>BDSC_16799, BDSC_19894, BDSC_11664</td>
			<td>The stocks of Drosophila melanogaster mutant</td>
		</tr>
		<tr>
			<td>Electric blast drying oven</td>
			<td>Tianjin Taiste Instruments</td>
			<td>101-0AB</td>
			<td>For coating slides and paraffin embedding</td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>Sigma-Aldrich</td>
			<td>230251</td>
			<td>Hematoxylin-Eosin Staining</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sinopharm Chemical Reagent at Beijing</td>
			<td>100092680</td>
			<td>Paraffin Embedding, Hematoxylin-Eosin Staining, Immunohistochemistry, In-situ Hybridization and Periodic Acid-Silver Methenamine Staining</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sinopharm Chemical Reagent at Beijing</td>
			<td>10010328</td>
			<td>Coating Slides</td>
		</tr>
		<tr>
			<td>Gold chloride</td>
			<td>Sigma-Aldrich</td>
			<td>379948</td>
			<td>Periodic Acid-Silver Methenamine Staining</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Sigma-Aldrich</td>
			<td>H3136</td>
			<td>Hematoxylin-Eosin Staining</td>
		</tr>
		<tr>
			<td>High Pure PCR Product Purification Kit</td>
			<td>Roche</td>
			<td>11732668001</td>
			<td>For purification of PCR products</td>
		</tr>
		<tr>
			<td>Intelligent constant temperature and humidity box</td>
			<td>Ningbo Jiangnan Instruments</td>
			<td>HWS</td>
			<td>For fly maintenance</td>
		</tr>
		<tr>
			<td>LE Agarose</td>
			<td>HyAgarose</td>
			<td>14190108029</td>
			<td>Pre-embedding</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sinopharm Chemical Reagent at Beijing</td>
			<td>10014108</td>
			<td>Drosophila Embryo Collection</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>ZEISS</td>
			<td>Observer.A1</td>
			<td>Hematoxylin-Eosin Staining, Immunohistochemistry, In-situ Hybridization and Periodic Acid-Silver Methenamine Staining</td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>MeVid Labware Manufacturing</td>
			<td>P105-2001</td>
			<td>Coating Slides</td>
		</tr>
		<tr>
			<td>Neutral Gum</td>
			<td>Sinopharm Chemical Reagent at Beijing</td>
			<td>10004160</td>
			<td>Hematoxylin-Eosin Staining</td>
		</tr>
		<tr>
			<td>N-heptane</td>
			<td>Sinopharm Chemical Reagent at Beijing</td>
			<td>40026768</td>
			<td>Drosophila Embryo Collection</td>
		</tr>
		<tr>
			<td>Paraffin slicer</td>
			<td>Huahai science instrument</td>
			<td>HH-2508III</td>
			<td>In-situ Hybridization</td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Sinopharm Chemical Reagent at Beijing</td>
			<td>69019461</td>
			<td>Paraffin Embedding</td>
		</tr>
		<tr>
			<td>pH/mV Meter</td>
			<td>Sartorius</td>
			<td>PB-10</td>
			<td>For determing the pH value of a solution</td>
		</tr>
		<tr>
			<td>Silver nitrate</td>
			<td>Sinopharm Chemical Reagent at Beijing</td>
			<td>10018461</td>
			<td>Periodic Acid-Silver Methenamine Staining</td>
		</tr>
		<tr>
			<td>Ultrapure water meter</td>
			<td>Thermo</td>
			<td>AFXI-0501-P</td>
			<td>In-situ Hybridization</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sinopharm Chemical Reagent at Beijing</td>
			<td>10023418</td>
			<td>Paraffin Embedding</td>
		</tr>
	</tbody>
</table>

a protocol for immunohistochemistry and rna in-situ distribution within early drosophila embryo

钙诱导钙释放信号传导（CICR）在许多生物过程中起着关键作用。从细胞增殖和凋亡，发育和衰老到神经元突触可塑性和再生的每一个细胞活动都与Ryanodine受体（RyRs）有关。尽管钙信号传导的重要性，但其功能在早期发育中的确切机制尚不清楚。作为一种妊娠期较短的生物体，黑 腹果蝇 的胚胎是研究CICR相关蛋白及其调节剂在发育过程中的分布和定位的主要研究对象。然而，由于它们的富含脂质的胚胎和富含几丁质的绒毛膜，它们的实用性受到将胚胎安装在玻璃表面上的难度的限制。在这项工作中，我们引入了一种实用的方案，可显着增强 果蝇 胚胎在载玻片上的附着力，并详细介绍了成功组织化学，免疫组织化学和 原位 杂交的方法。铬明矾明胶造布法和胚胎预包埋法大大提高了 果蝇 胚胎蛋白和RNA表达研究的产量。为了证明这种方法，我们研究了DmFKBP12 / Calstabin，这是黑 腹果蝇早期胚胎发育期间RyR的着名调节因子。我们早在合胞胚芽孢子阶段就鉴定了 DmFKBP12 ，并报告了 DmFKBP12 在发育过程中的动态表达模式：最初作为合胞体胚层中均匀分布的蛋白质，然后在细胞胚泡期间初步定位到皮层的基底层，然后在早期原肠胚形成的三宝石层阶段分布在原始神经元和消化结构中。这种分布可以解释RyR在起源于这些层的重要器官系统中的关键作用：食管下和食管上神经节，腹侧神经系统和肌肉骨骼系统。

这些数字描述了用于克服将高脂质和含甲壳素的绒毛膜 果蝇 胚胎（表1）附着到载玻片表面进行检查和实验的挑战的方案。利用 图1所示的铬明矾明胶载玻片包衣方法，我们增强了 果蝇 胚胎在载玻片表面的附着力，而 图2 所示的胚胎预包埋方法可以有效地检查蛋白质和RNA DmFKBP12 / Calstabin在所有四个早期发育阶段（即合?...

Watch this Scientific Journal Video about A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo at JoVE.com

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

在这里，我们描述了果 蝇 胚胎蛋白和RNA的检测和定位方案，从收集到预包埋和包埋，免疫染色和mRNA 原位 杂交。

早期果蝇胚胎内免疫组织化学和RNA原位分布的方案

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and ...

作为一种妊娠期较短的生物体，黑腹果蝇的胚胎是研究蛋白质及其调节剂在发育过程中的分布和定位的主要研究对象。然而，由于它们的富含脂质的胚胎和富含几丁质的绒毛膜，它们的实用性受到将胚胎安装在玻璃表面上的难度的限制。在这项工作中，我们介绍了一种实用的方法，该方法显着增强了果蝇胚胎在载玻片上的附着力，并详细介绍了我们成功组织化学，免疫组织化学和原位杂交的方法。在执行此处所示的步骤之前，应用洗涤剂清洗载玻片，然后在自来水和蒸馏水中冲洗，然后放入烤箱中晾干。干燥的载玻片应在重铬酸钾中轻轻浸泡24小时，然后冲洗并再次干燥，然后置于95%乙醇中至少两小时。在此步骤中，您可以根据我们的方法准备进一步的载玻片或铬明矾明胶。然后，明胶可以在使用前储存在60度的洗澡水中。要涂覆载玻片，请将少量明胶滴到载玻片的一个边缘。然后使用另一个载玻片缓慢而均匀地将明胶拖过要涂覆的载玻片表面。这将创建一个薄而均匀的图层。在放入架子或干燥设备中储存在烤箱中以允许再完全干燥过夜或48小时之前。使用葡萄汁琼脂平板收集胚胎，一旦准备好，就可以取出葡萄汁琼脂平板。用PBS填充板，并用软刷轻轻刷子，以去除任何附着的胚胎。将含有PBS的胚胎取出到1.5毫升离心管中，并在冰上休息。一旦胚胎在冰上固定，轻轻取出上清液PBS。注意不要打扰管底部的胚胎。向样品中加入1 mL 50%漂白剂，轻轻摇匀两分钟。通过过滤去除漂白剂，并在PBS中洗涤胚胎三次。准备正庚烷。准备就绪后，通过在溶液中轻轻摇动滤纸，将滤纸中的胚胎洗涤成正庚烷。让胚胎沉降在正庚烷板的底部，并将该正庚烷收集到新的1.5毫升离心管中。向管中加入一毫升甲醇，让溶液沉淀，然后吸出正庚烷甲醇。在PBS中洗涤胚胎三到五次以除去残留的甲醇。取出这个额外的PBS，加入Bouin的固定剂，静置30到60分钟，以固定胚胎。取出Bouin的固定剂，并在PBS中再次洗涤胚胎三次。在PBS中制备1.2%琼脂糖，并在60度水浴中保持温暖，然后通过轻轻吸出已储存的额外PBS来准备胚胎以进行嵌入。小心不要打扰底部的胚胎，然后吸出溶液底部的胚胎塞，轻轻放入瓶盖模具中。在加入1.2%琼脂糖凝胶之前，从该模具中吸取任何额外的PBS。准备PBS溶液以储存这些琼脂糖插头。一旦琼脂糖凝固，就可以将单个插头移动到此PBS中进行存储。在这里，我们展示了顺序脱水过程。每次孵育15分钟。然后从100%乙醇中取出，并在乙醇和二甲苯的一对一溶液中再固定15分钟。从这种一对一的溶液中除去后，在纯二甲苯中除去一分钟。现在，组织样品已准备好移动到加热的一对一二甲苯石蜡溶液中。静置30分钟，然后最终嵌入100%石蜡中。移至100%石蜡中的第一个，休息两个小时。在两个和三个石蜡中重复此过程。样品现在可以移动到模具中，模具中装满蜡以完成包埋过程。果蝇胚胎的嵌入。将准备好的果蝇胚胎石蜡切片放入60度烤箱中15分钟。将它们置于100%二甲苯溶液中。然后我们开始补液。将幻灯片放在每个溶液中三分钟。一旦载玻片在蒸馏水中，放入苏木精溶液中两分钟。完成后，确保从载玻片中排出尽可能多的溶液。将载玻片用蒸馏水洗涤后，将载玻片在曙红中染色一分钟。最后，从曙红中取出载玻片，在蒸馏水中洗涤。一旦这张幻灯片干净，我们现在开始最后的脱水过程。最后放入100%二甲苯中五分钟。在两种100%二甲苯的新溶液中重复该过程，并沿着载玻片的一个边缘滴下少量中性胶。取下此盖玻片，轻轻地放在滑盖的一个边缘。首先通过放入100%二甲苯来脱蜡载玻片。一旦载玻片从蒸馏水中取出，应用1%的元素酸溶液覆盖它们。然后，我们丢弃多余的溶液，用蒸馏水冲洗。通过将载玻片直接放入预热的溶液中。一旦完成，我们用蒸馏水冲洗载玻片，然后用0.2%氯化金着色溶液一到两分钟。接下来，我们用3%硫代硫酸钠用蒸馏水固定组织三分钟。然后用苏木精复染色。复染后，可以用任何一管蒸馏水清洗污渍，如图所示。然后我们进入脱水过程。然后将载玻片直接放入100%二甲苯中，然后在载玻片的角落涂抹少量中性口香糖。用盖玻片轻轻盖住。像以前一样，通过脱蜡和二甲苯开始制备载玻片。然后在一对一的二甲苯乙醇中。一旦载玻片脱蜡，我们开始重新水化它们的组织，然后最终在PBS中。将载玻片置于甲醇制成的0.3%过氧化氢中10分钟。然后用20 mL的目标检索溶液覆盖载玻片。然后可以将载玻片放入染色盒中。将染色盒放入蒸笼中，确保没有溶液逸出，载玻片仍完全覆盖。请注意，污渍盒盖不应完全关闭，以免水蒸气在蒸气过程中逸出。然后在PBS-T中轻轻冲洗三次。丢弃多余的溶液并重复该过程，这次是使用PBS。接下来，我们将载玻片在过氧化物块中孵育5至10分钟，然后在PBS-T中再次冲洗几次，然后在PBS中冲洗。准备抗体并直接滴到组织上。然后可以将这些组织孵育或在室温下孵育四小时。二抗应在室温下孵育90分钟，用5%DAB染色2至10分钟。获得适当的颜色后，在蒸馏水中冲洗，然后在苏木精中复染。在这种苏木精染色后，载玻片可以依次脱水。将载玻片放入100%二甲苯中，浸泡三分钟。然后将少量中性口香糖滴到载玻片的一个边缘。放置口香糖后，取下载玻片盖，从载玻片的一个边缘轻轻放置。将一微克纯化的线性化模板DNA加入无菌无RNase反应瓶中。然后加入足够无菌的无RNase DEPC处理的双蒸馏水，使样品总体积为13微升。然后短暂离心，在37摄氏度下孵育两个小时。对于RNase保护实验，我们建议用两微升无RNase的Dnase-1孵育15分钟以去除模板DNA。孵育步骤完成后，通过在pH值为8时加入两微升0.2摩尔EDTA来停止反应。在本文显示的步骤之前，根据我们之前演示的实验方案，载玻片已脱蜡并重新水合。补液后，将载玻片以每升PBS0.01摩尔的pH值孵育5分钟，每次5分钟，以从组织中除去固定溶液。在PBS中冲洗后，我们接下来用每升100毫摩尔甘氨酸溶液冲洗五分钟两次，以从组织中除去游离的醛基团。完成此步骤后，用 PBS 冲洗载玻片五分钟。接下来，在含有0.3%triton X-100的PBS中孵育15分钟，然后冲洗，然后再次孵育每微升蛋白酶K.接下来，用4%多聚甲醛和PBS在pH值为7.2时固定载玻片三分钟。然后，在用0.1摩尔三乙醇胺缓冲液pH 8孵育之前洗涤载玻片，其中含有0.25%乙酸酐。您可以选择在此阶段倾斜盒子，以确保解决方案完全覆盖。通过将20%甘油或DEPC水添加到干杂交盒的底部来制备该盒。接下来，在20微升的预杂交溶液中覆盖载玻片。鲑鱼的精子DNA浓度为每毫升100微克到每张载玻片。然后在42摄氏度下孵育四个小时。这是在预先准备好的加湿盒中完成的，以确保溶液在此过程中不会干燥。在杂交后阶段，从样品中完全冲洗杂交溶液非常重要。这可以在37度下连续冲洗15分钟，使用四倍浓度SSC，两倍浓度一倍浓度，0.5倍浓度和0.1倍浓度SSC溶液。每个在37摄氏度下孵育15分钟。完成后，用PBS洗涤三次，每次五分钟，然后加入洗涤缓冲液。在进行后续步骤之前，应冲洗洗涤缓冲液一分钟，确保在加入下一个溶液之前，洗涤缓冲液从载玻片中完全干燥。向每张载玻片中加入100毫升阻塞溶液，并将载玻片孵育30分钟。最好在无光的环境中进行，以确保良好的染色效果。接下来，将载玻片在20 mL抗体溶液中再次孵育30分钟。将抗体溶液直接涂抹在载玻片上，以完全覆盖您的组织。孵育完成后，施用100毫升洗涤缓冲液。在无光照条件下孵育15分钟，以除去未结合的抗体偶联物。取出洗涤缓冲液。接下来，应用20毫升检测缓冲液，并使其孵育约4分钟。四分钟后，取出检测缓冲区。干燥载玻片以确保没有额外的溶液，并加入10毫升新鲜制备的彩色底物溶液。该反应在几分钟内开始，但在16小时后完成。因此，允许在无光照的条件下孵育一段时间。一旦检测到条带的所需斑点，用50毫升TE缓冲液洗涤载玻片以停止反应。然后完成我们之前展示的中性口香糖脱水过程和安装过程。图1和图2展示了铬铝明胶包衣方法在发育中的果蝇胚胎中用于石蜡切片以及FKBP12和DmFKBP12表达谱的包埋方法。图三显示了在合胞细胞胚芽孢子阶段检测到的抗体FKBP12的蛋白质分布。从这些组织化学结果中可以看出，FKBP12表达在合胞胚芽孢子虫中的极化程度较低，然后开始在滋养外胚层上皮下方的基底膜内定位于梯度，在前极和后极中发现更多的表达。图四，遵循果蝇胚胎的早期和晚期气体关系阶段，显示FKBP12蛋白被限制在原始胚胎组织的某些结构成分上。细胞外分布少于在早期和晚期胃胚胎阶段观察到的分布。有趣的是，上述动态蛋白质分布并不伴随着FKBP12的MRI水平的相应变化，如图五所示。表明蛋白质表达是仍然未知的转录后分子机制的结果。Ryanodine受体介导的钙信号传导是脊椎动物和无脊椎动物的许多生理和病理过程的基本途径。已知该基因的突变会导致许多生理紊乱，导致疾病或早期心脏死亡。然而，该基因在早期果蝇发育中的作用一直难以研究。作为胚胎的中转成分，富含脂质的胚胎和富含几丁质的绒毛膜，使得研究果蝇早期发育中的蛋白质和胚胎表达变得困难。本文中描述的载玻片包衣，胚胎包埋和免疫组织化学技术使我们能够详细研究ryanodine受体蛋白和mRNA的动态分布。我们分享这些技术，并希望它们能让其他人在果蝇的早期发育阶段研究其他蛋白质。

Research

JoVE Journal

Developmental Biology

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

了解早期器官使用简体<em&gt;原位</em&gt;杂交协议<em&gt;爪</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

科研

发育生物学

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

协议获得合子和体细胞胚胎研究早期胚胎发育的调控中的示范豆科<em&gt;苜蓿</em

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by ...

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

中等规模的制备<em&gt;果蝇</em&gt;蛋白质组学实验胚胎提取物

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, ...

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

全贴装荧光原位杂交和免疫荧光法在斑马鱼中 Multiciliated 细胞的可视化

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

家兔模型最佳胚胎阶段微创胚胎移植与胚胎玻璃化

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

嗜酸性粒植物视网膜免疫组织化学、西方分析和 Rna 分离的解剖

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

果蝇胚胎肌肉收缩的实时成像和分析

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

解冻、培养和冷冻保存的嗜酸性粒细胞系

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

一种直接而简单的方法，用于评估从胚胎到成人的果蝇黑色素的生存能力

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

为早期小鼠胚胎测序准备小型RNA库

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...