全山原位杂交E8.5，E11.5小鼠胚胎
Summary
这整个坐骑<em>原位</em>杂交协议的讨论，以确保在E8.5 – E11.5日龄小鼠胚胎的基因表达研究的重复性高品质的成果的关键步骤。
Abstract
整装原位杂交，是一个非常翔实的办法确定胚胎中的基因表达模式。 原位杂交程序冗长，技术上与多个重要步骤，集体作出贡献的最终结果的质量要求，该协议详细描述了优化探针标记和性能的几个关键质量控制步骤。总的来说，我们的协议提供了详细的说明必要的关键步骤，可重复获得高质量的结果。首先，我们描述了一代digoxygenin（DIG）标记的RNA探针通过在体外经PCR产生的DNA模板转录。我们描述了三个关键的质量控制检测，以确定的数量，完整性和辛标记探针的具体活动。这些步骤是重要的生成足够的灵敏度探测器检测内源性的mRNA在整个小鼠胚胎。此外，我们描述的固定和储存E8.5 – E11.5日龄小鼠胚胎原位杂交的方法。然后，我们描述了详细的方法有限蛋白酶K消化杂交条件的细节，杂交后洗涤和核糖核酸酶处理，除去非特异性探针杂交的水化胚胎。 AP -共轭抗体用于可视化探针，并揭示了内源性转录的表达模式。代表实验成功和典型的不理想的实验结果显示。
Protocol
1。通过体外转录的Riboprobe代 准备在体外转录模板的PCR产物 。 噬菌体在其5'端转录启动子序列设计PCR引物。 注：启动子序列的意义链的PCR引物5'端，将抄录感探头，并添加到启动子序列的反义PCR引物5'端，将合成的反义探针1-3。我们并没有发现任何差异之间的模板，其中包含只有核心启动子序列与模板包含核心启动子加上额外的5'序…
Discussion
在本议定书中所描述的方法已经适应从不同来源和整装E8.5 – E11.5日龄小鼠胚胎进行了优化。整个装载在脊椎动物胚胎原位杂交方法最早出现在20世纪90年代初2,5-12。该协议被改编，主要是从非洲爪蟾胚胎 7,8以及鼠标 2,11开发的方法。在我们的协议中，很大的重点放在探头严格的质量评估。仔细注意产生高品质和高比活性的RNA探针在这个协议中最早的步骤确定将…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由国立卫生研究院拨款R21MH082360（BGC）和R01HD056315（NRM）以及佐治亚大学的支持。
Materials
| Name | Type | Company | Catalogue number | Comments |
|---|---|---|---|---|
| Digoxigenin-11-uridine-5′-triphosphate | Reagent | Roche | 11209256910 | |
| Ribonucleoside Triphosphate Set | Reagent | Roche | 11277057001 | |
| Quick Spin Columns for radiolabeled RNA purification | Supply | Roche | 11274015001 | |
| T7 RNA polymerase | Reagent | Roche | 10881767001 | |
| SP6 RNA polymerase | Reagent | Roche | 10810274001 | |
| T3 RNA polymerase | Reagent | Roche | 11031163001 | |
| CTP, [α-32P]- 800Ci/mmol 10mCi/ml , 250 μCi | Reagent | Perkin Elmer | BLU008X250UC | |
| DNase I recombinant, RNase-free | Reagent | Roche | 4716728001 | |
| Urea,Colorless-to-white Crystals or Crystalline Powder | Reagent | Fisher | BP169-500 | |
| Gel loading buffer | Reagent | Ambion | AM8547 | |
| Hybond-N+, Amersham | Supply | GE Healthcare | RPN82B | |
| UV Stratalinker 2400 | Equipment | Stratagene | ||
| Diethyl pyrocarbonate | Reagent | Sigma | D5758-100ML | |
| Heparin sodium | Reagent | Acros | 41121-0010 | |
| hydrogen peroxide 30% in water | Reagent | Fisher scientific | BP2633-500 | |
| Gluteraldehyde, 8% EM grade | Reagent | Polysciences | 0/710 | Use with caution according to manufacture’s instructions |
| Blocking reagent | Reagent | Roche | 11096176001 | Make into 5% stock and store at -20° C |
| Proteinase K | Reagent | Roche | 3115852001 | |
| Rnase A | Reagent | Roche | 10109142001 | Make as 10 mg/ml stock and store at -20° C. |
| Rnase T1 | Reagent | Roche | 10109193001 | |
| Ultrapure Formamide | Reagent | Invitrogen | 15515-026 | |
| Levamisole hydrochloride | Reagent | ICN Biomedicals. Inc | 155228 | |
| Ribonucleic acid from torula yeast,Type VI | Reagent | Sigma | R6625 | phenol/chloroform extracted several times and precipitated, resuspended in DEPC-dH2O and stored at -20° C |
| Anti-Digoxigenin-AP Fab fragments | Reagent | Roche | 11093274910 | |
| BM Purple AP Substrate, precipitating. Ready-to-use solution. | Reagent | Roche | 11 442 074 001 | |
| 4mL, Clear, Closed Top, Storage Vial Convenience Kit | Supply | National scientific | B7800-2 | |
| Shake N Bake hybridization oven | Equipment | Boekel Scientific | 136400 | |
| Biopsy Sure-Tek | Supply | Fisherbrand | 15-200-402C | |
| Paraplast Plus tissue embedding medium | Reagent | Fisherbrand | 23-021-400 | |
| Peel-A-Way disposable plastic tissue embedding molds | Supply | Polysciences | 18646A | |
| Colorfrost Plus Microscope slides | Supply | Fisherbrand | 12-550-17 | |
| Nuclear Fast Red | Reagent | Sigma | N8002 | |
| Cytoseal 60 | Reagent | Richard-Allan Scientific | 8310-16 |
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Tags
Whole MountIn Situ HybridizationGene Expression PatternsEmbryosProbe LabelingQuality ControlDIG-labeled RNA ProbesIn Vitro TranscriptionPCRSensitivityEndogenous MRNAFixationStorageProteinase K DigestionHybridization ConditionsPost-hybridization WashesRNase TreatmentNon-specific Probe HybridizationAP-conjugated Antibody
Cite This Article
Wei, Q., Manley, N. R., Condie, B. G. Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos. J. Vis. Exp. (56), e2797, doi:10.3791/2797 (2011).