早期果蝇胚胎内免疫组织化学和RNA原位分布的方案
1National Engineering Laboratory for Resource Development of Endangered Crude Drugs in Northwest of China/CGDB,Shaanxi Normal University College of Life Sciences, 2Shaanxi Key Laboratory of Ischemic Cardiovascular Disease, Shaanxi Key Laboratory of Brain disorders, Institute of Basic & Translational Medicine,Xi’an Medical University, 3Ohio State University College of Medicine, 4Hematology-Oncology Division, Department of Medicine, Beth Israel Deaconess Medical Center,Harvard Medical School, 5University of Pennsylvania ULAR, 6University of Maryland School of Medicine, 7Columbia University Medical Center
Summary
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。
Abstract
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。
Introduction
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。
Protocol
1. 葡萄汁琼脂平板的制备 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。 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。 2. 涂布载玻片 用洗涤剂预处理载玻片。用自来水清洗载玻片3?…
Representative Results
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?…
Discussion
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?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
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）。
Materials
| -20°C Refrigerator | Meiling Biology &Medical | DW-YL270 | Used for regent storage |
| -80°C Ultra low temperature refrigerator | Thermo | Forma 90 Series | Used for regent storage |
| Agar | Sigma-Aldrich | WXBB6360V | Preparation of grape juice agar plates |
| Anti-Digoxigenin-AP, Fab fragments | Roche | 11093274910 | For the detection of digoxigenin-labeled compound |
| Biochemical incubator | Shanghai Bluepard Instruments | LRH-250 | In-situ Hybridization |
| Bouin's solution | Sinopharm Chemical Reagent at Beijing | 69945460 | Drosophila Embryo Embedding |
| Centrifuge | Eppendorf | 540BH07808 | In-situ Hybridization |
| Centrifuge tube | Denville | C-2170 | Drosophila Embryo Collection |
| Chrome Alum | Sinopharm Chemical Reagent at Beijing | 10001018 | Coating Slides |
| Constant temperature water bath | Jintan Henfeng Instruments | KW-1000DC | Hematoxylin-Eosin Staining, Immunohistochemistry, In-situ Hybridization and Periodic Acid-Silver Methenamine Staining |
| Dako REAL EnVision Detection System | Dako | K5007 | In immunohistochemical reaction or in situ hybridization reaction, it binds to the primary antigen antibody, and the target is labeled by staining. |
| DEPC | Sigma-Aldrich | D5758 | In-situ Hybridization |
| DIG RNA Labeling Kit | Roche | 11093274910 | RNA labeling with diagoxigenin-UTP by in vitro transcription with SP6 and T7 RNA polymerase |
| Drosophila melanogaster | Bloomington Stock Center | BDSC_16799, BDSC_19894, BDSC_11664 | The stocks of Drosophila melanogaster mutant |
| Electric blast drying oven | Tianjin Taiste Instruments | 101-0AB | For coating slides and paraffin embedding |
| Eosin | Sigma-Aldrich | 230251 | Hematoxylin-Eosin Staining |
| Ethanol | Sinopharm Chemical Reagent at Beijing | 100092680 | Paraffin Embedding, Hematoxylin-Eosin Staining, Immunohistochemistry, In-situ Hybridization and Periodic Acid-Silver Methenamine Staining |
| Gelatin | Sinopharm Chemical Reagent at Beijing | 10010328 | Coating Slides |
| Gold chloride | Sigma-Aldrich | 379948 | Periodic Acid-Silver Methenamine Staining |
| Hematoxylin | Sigma-Aldrich | H3136 | Hematoxylin-Eosin Staining |
| High Pure PCR Product Purification Kit | Roche | 11732668001 | For purification of PCR products |
| Intelligent constant temperature and humidity box | Ningbo Jiangnan Instruments | HWS | For fly maintenance |
| LE Agarose | HyAgarose | 14190108029 | Pre-embedding |
| Methanol | Sinopharm Chemical Reagent at Beijing | 10014108 | Drosophila Embryo Collection |
| Microscope | ZEISS | Observer.A1 | Hematoxylin-Eosin Staining, Immunohistochemistry, In-situ Hybridization and Periodic Acid-Silver Methenamine Staining |
| Microscope Slides | MeVid Labware Manufacturing | P105-2001 | Coating Slides |
| Neutral Gum | Sinopharm Chemical Reagent at Beijing | 10004160 | Hematoxylin-Eosin Staining |
| N-heptane | Sinopharm Chemical Reagent at Beijing | 40026768 | Drosophila Embryo Collection |
| Paraffin slicer | Huahai science instrument | HH-2508III | In-situ Hybridization |
| Paraffin | Sinopharm Chemical Reagent at Beijing | 69019461 | Paraffin Embedding |
| pH/mV Meter | Sartorius | PB-10 | For determing the pH value of a solution |
| Silver nitrate | Sinopharm Chemical Reagent at Beijing | 10018461 | Periodic Acid-Silver Methenamine Staining |
| Ultrapure water meter | Thermo | AFXI-0501-P | In-situ Hybridization |
| Xylene | Sinopharm Chemical Reagent at Beijing | 10023418 | Paraffin Embedding |
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Zhang, W., Lei, X., Zhou, X., He, B., Xiao, L., Yue, H., Wang, S., Sun, Y., Wu, Y., Wang, L., Ghartey-Kwansah, G., Jones, O. D., Bryant, J. L., Xu, M., Ma, J., Xu, X. A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo. J. Vis. Exp. (183), e61776, doi:10.3791/61776 (2022).