整个山<em>原位</em>对腹足软体动物杂交方法<em>静水椎实螺</em
Summary
The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.
Abstract
整体原位杂交（WMISH）是一种技术，其允许核酸分子（通常的mRNA）的“整装'组织制剂内的空间分辨率，或发育阶段（例如胚胎或幼虫）的兴趣。 WMISH是非常强大的，因为它可以显著有助于复杂的后生动物基因组的功能特性，正在成为更多的瓶颈与下一代序列数据的泛滥是一个挑战。尽管该技术多的时间，通常需要以优化固有为新颖模型系统WMISH实验的各种参数的概念简单;在组织类型和发育阶段之间的细胞和生化特性的细微差异意味着一个单WMISH方法可能并不适用于所有的情况。我们已经制定了一套产生一致的和重新出现的腹模型静水椎实螺 WMISH方法明确WMISH信号一系列的基因，并在所有发展阶段。这些方法包括未知实足年龄的幼虫到一个个体发育窗口分配，有效地除去胚胎和幼虫从它们的卵胶囊剂，合适的蛋白酶K处理的每个个体发育窗口的应用，和杂交，杂交后和免疫检测脚步。这些方法提供从其中对于给定的RNA转录所得到的信号可以是与探测器专用的调整（主要探针浓度和杂交温度）进一步精制的基础。
Introduction
软体动物是一组持有学科的广泛多样化的利益动物。尽管他们的形态多样性1，物种丰富度（仅次于节肢动物的种类数2项）和相关性，以广泛的商业3，4医学和科学问题5-8，也有可以称得上相对较少的种软体动物既装备精良的科学模型，容易在实验室环境维护。即多使用学科，如神经生物学9，生态毒理学10和最近的进化生物学11,12一种软体动物，是静水椎实螺，主要是它的广泛分布和极端易于维护的，因为。尽管它作为一种“模式”有机体的知名度和其悠久的历史使用通过提供给L.发育生物学家13-19，分子工具的范围和功率stagnALIS科学界位于远远落后于更为传统的动物模型（ 果蝇 ，小鼠，海胆，线虫）。
我们的愿望，开发椎实螺的分子模型在指导蛋壳形成的分子机制的兴趣茎。这促使我们缩小一组技术，将允许对椎实螺的发育过程中的基因表达的有效的，一致的和敏感的可视化。 整体原位杂交（WMISH）被广泛用于各种模式生物的和已经使用了超过40年20。在它的不同的形式，ISH可以采用在空间上定位于染色体，rRNA基因，mRNA和微RNA的特异位点。
面临的挑战之一，我们需要解决炼制WMISH方法L.之前stagnalis是温和而有效地提取胚胎和从T不同阶段的幼虫的问题他在它们所沉积的蛋胶囊。该提取或“解封”，需要被有效地实现，以收集足够的物质用于现场试验给出，而在同一时间保持形态和细胞完整性。虽然其他模式生物，也经历了发展封装，在我们手中没有任何报道这些物种的方法可以成功地使用L. stagnalis。
因此该方法的总体目标是：以提取L. stagnalis从它们在高通量胶囊，胚胎和幼虫以施加优化WMISH信号，以提供满意的WMISHsignals用于成像准备胚胎和幼虫预杂交处理。
Protocol
注：以下步骤概括我们的方法对L.的胚胎和幼体阶段进行的现场试验stagnalis。当一个步骤涉及使用危险化学品这是由词'小心'和所有适当的安全程序的表示应采用。在补充文件1提供链接代表MSDS表的危险化学品。所有试剂配方在补充文件2提供。 1.解封设备的组装要做到这一点，连接20ml的一次性注射器，以硅管（1毫米?…
Representative Results
在图3所示的代表WMISH染色图案用上面描述的技术产生的，并反映了所涉及的范围内的分子过程，从外壳形成的基因的各种空间表达模式的（ 新基因1，2，3和4），细胞-细胞信号（DPP）在一系列发育阶段的转录调控（ 短尾 ）。虽然我们还没有量化的这些基因，我们期望他们也表达水平变化显著，这表明我们的方法可以对各种各样的处…
Discussion
这里所描述的方法允许RNA转录物与静水椎实螺的所有发育阶段内想必改变的表达水平的高效率的可视化。为了从它们的胶囊除去胚胎和幼虫我们造模报道其他encapsulated-各种化学，渗透压休克和物理处理的开发模式生物。然而，在我们的手中，我们在这里描述的方法是唯一的高通量技术，消除了艰难的囊膜不破坏胚胎和幼虫。以下解封装，该材料可以被存储，或与蛋白酶K的阶段具体?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作是通过DFG项目＃JA2108 / 2-1资金支持DJJ。
Materials
| Featherweight forceps | Ehlert & Partner | #4181119 | |
| Silicon tubing | Glasgerätebau OCHS GmbH | 760070 | |
| Glass capillaries | Hilgenberg | 1403547 | |
| 12 well tissue culture dishes | Carl Roth | CE55.1 | |
| 37% Formaldehyde | Carl Roth | P733.1 | CAUTION – May cause cancer. Toxic by inhalation, in contact with skin and if swallowed. Toxic: danger of very serious irreversible effects through inhalation, in contact with skin and if swallowed. |
| Ethylenediamine tetraacetic acid | Carl Roth | CN06.3 | CAUTION – CAUSES EYE IRRITATION. MAY CAUSE RESPIRATORY TRACT AND SKIN IRRITATION. Avoid breathing dust. Avoid contact with eyes, skin and clothing. Use only with adequate ventilation |
| Magnesium Chloride | Carl Roth | 2189.1 | |
| Tween-20 | Carl Roth | 9127.1 | CAUTION – May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if absorbed through skin. May cause skin irritation. May cause eye irritation. May be harmful if swallowed. |
| Sodium Chloride | Carl Roth | 3957.1 | |
| Ficoll type 400 | Carl Roth | CN90.1 | |
| polyvinylpyrrolidone K30 (MW 40) | Carl Roth | 4607.1 | CAUTION – May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if absorbed through skin. May cause skin irritation. May cause eye irritation. May be harmful if swallowed. |
| Nuclease freeBovine Serum Albumin | Carl Roth | 8895.1 | |
| Salmon sperm | Carl Roth | 5434.2 | |
| Heparin | Carl Roth | 7692.1 | CAUTION – ADVERSE EFFECTS INCLUDE HEMORRHAGE, LOCAL IRRITATION. POSSIBLE ALLERGIC REACTION IF INHALED, INGESTED/CONTACTED. EYES/SKIN/RESPIRATORY TRACT IRRITANT. POSSIBLE HYPERSENSITIZATION. DURING PREGNANCY HAS BEEN REPORTED TO INCREASE RISK OF STILLBIRTH |
| Proteinase-K | Carl Roth | 7528.1 | |
| Glycine | Carl Roth | 3790.2 | |
| Deionised formamide | Carl Roth | P040.1 | CAUTION – Irritating to eyes and skin. May be harmful by inhalation, in contact with skin and if swallowed. May cause harm to the unborn child. Hygroscopic. |
| Standard formamide | Carl Roth | 6749.3 | CAUTION – Irritating to eyes and skin. May be harmful by inhalation, in contact with skin and if swallowed. May cause harm to the unborn child. Hygroscopic. |
| Triethanolamine | Carl Roth | 6300.1 | CAUTION – Avoid breathing vapor or mist. Avoid contact with eyes. Avoid prolonged or repeated contact with skin. Wash thoroughly after handling. |
| Acetic anhydride | Carl Roth | 4483.1 | CAUTION – CAUSES SEVERE SKIN AND EYE BURNS. REACTS VIOLENTLY WITH WATER. HARMFUL IF SWALLOWED. VAPOR IRRITATING TO THE EYES AND RESPIRATORY TRACT |
| Maleic acid | Carl Roth | K304.2 | CAUTION – Very hazardous in case of eye contact (irritant), of ingestion, . Hazardous in case of skin contact (irritant), of inhalation (lung irritant). Slightly hazardous in case of skin contact (permeator). Corrosive to eyes and skin. |
| Benzyl benzoate | Sigma | B6630-250ML | CAUTION – May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if absorbed through skin. May cause skin irritation. May cause eye irritation. Harmful if swallowed. |
| Benzyl alcohol | Sigma | 10,800-6 | CAUTION – Harmful if swallowed. Harmful if inhaled. Causes serious eye irritation. |
| Glycerol | Carl Roth | 3783.1 | |
| Blocking powder | Roche | 11096176001 | |
| Anti DIG Fab fragments AP conjugated | Roche | 11093274910 | |
| Tris-HCl | Carl Roth | 9090.3 | |
| 4-Nitro blue tetrazolium chloride in dimethylformamide | Carl Roth | 4421.3 | CAUTION – May cause harm to the unborn child. Harmful by inhalation and in contact with skin. Irritating to eyes. |
| 5-bromo-4-chloro-3-indolyl-phosphate | Carl Roth | A155.3 | CAUTION – Potentially harmful if ingested. Do not get on skin, in eyes, or on clothing. Potential skin and eye irritant. |
| N-acetyl cysteine | Carl Roth | 4126.1 | |
| Dithiothreitol | Carl Roth | 6908.1 | CAUTION – May cause eye and skin irritation. May cause respiratory and digestive tract irritation. The toxicological properties of this material have not been fully investigated. |
| Tergitol | Sigma | NP40S | CAUTION – May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if absorbed through skin. May cause skin irritation. May cause eye irritation. May be harmful if swallowed. |
| Sodium dodecyl sulphate | Carl Roth | CN30.3 | CAUTION – Harmful if swallowed. Toxic in contact with skin. Causes skin irritation. Causes serious eye damage. May cause respiratory irritation. |
| Potassium Chloride | Carl Roth | 6781.1 | |
| di-Sodium hydrogen phosphate dihydrate (Na2HPO4.2H2O) | Carl Roth | 4984.1 | |
| Potassium dihydrogen phosphate (KH2PO4) | Carl Roth | 3904.1 | |
| Tri sodium citrate dihydrate (C6H5Na3O7.2H2O) | Carl Roth | 3580.1 | CAUTION – May cause eye, skin, and respiratory tract irritation. The toxicological properties of this material have not been fully investigated. |
| Mineral oil | Carl Roth | HP50.2 | |
| InSituPro-Vsi | Intavis | www.intavis.de/products/automated-ish-and-ihc |
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Citer Cet Article
Jackson, D. J., Herlitze, I., Hohagen, J. A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis. J. Vis. Exp. (109), e53968, doi:10.3791/53968 (2016).