结合免疫荧光和三维保存的间期核荧光原位杂交DNA研究在3D核组织的变化
Summary
在这里，我们描述了一个协议，用于同时检测组蛋白修饰免疫荧光法和DNA序列的DNA荧光原位杂交，然后通过的3D显微镜和分析（3D的免疫-DNA FISH）。
Abstract
荧光原位杂交DNA探针完好的三维保存的核三维激光共聚焦显微镜（3D DNA FISH）表示，最直接的方法来可视化基因位点的位置，染色体的次区域或整个地区的单个细胞。这种类型的分析提供洞察全球架构的细胞核，以及特定的基因组位点的行为和地区内的核空间。免疫荧光法，另一方面，允许核蛋白质（修饰组蛋白，组蛋白变体和改性剂，转录机械因素，核分舱室，等）的检测。免疫荧光和3D的DNA结合FISH的主要挑战是，一方面要保留的表位，以及由抗体检测的细胞核的3D架构上，另一方面，允许渗透的DNA探针来检测基因位点或染色体地区1-5。在这里，我们提供了一个协议，它结合了可视化的三维保存的核染色质修饰与基因位点在。
Introduction
表观遗传机制的发育和细胞类型特异的转录图谱的的触发建立和继承。在一个层面上，这涉及到调制的定义激活或沉默的基因组区域的染色质包装。在一个规模较大的基因组和核架构，全球3D组织也发挥了作用，在控制转录模式。因此，解剖这些表观基因的功能是必不可少的一个全面的了解基因是如何调控6-11。
结合免疫荧光和三维DNA鱼类提供了一个独特的机会，以补充分子生物学和生物化学的分析评估特定的DNA序列和/或细胞核内的蛋白质的相互作用/协会。此外，在全基因组高通量技术，如染色质免疫沉淀（ChIP-seq的）或染色体捕捉构象的深度测序（4C-seq技术，5C，加上HI-C）提供全球性的DAT一的细胞群12，免疫荧光/ DNA荧光原位杂交技术在单细胞水平上能够进行分析。
在这里，我们描述了一个协议，用于同时检测组蛋白修饰免疫荧光法和DNA序列的DNA荧光原位杂交，然后通过的3D显微镜和分析（3D免疫FISH）。此协议的优点是DNA和保存的蛋白质结构的组合的可视化。我们在这一领域的经验，使我们能够改进和简化现有的协议。虽然我们已经使用该协议来检测在淋巴细胞发生重组的DNA双链断裂，该方法可以应用到其他的蛋白质和其他细胞类型。
Protocol
1。 DNA探针标记荧光基团：：尼克翻译（约6小时） 清洁的BAC DNA（其制备由马克西制备）或质粒或PCR产物，所有的再悬浮在H 2 O中，可用于标记， 请注意 ，一个强大的FISH信号，探针应跨越至少10 kb的。 孵育DNA的RNA酶A中的30分钟，在37℃（ 表1中列出的所有试剂）。 在16°C孵育2小时缺口翻译反应（见表2和表3）。直接标记的替代方?…
Representative Results
DNA和免疫-FISH用于的Skok实验室，研究的过程中，V（D）J重组抗原受体位点在B和T淋巴细胞的发展与核组织的变化。上面详述的技术使我们ⅱ）到i）之间的测量距离的两个端部的轨迹（收缩）之间的测量距离的等位基因或基因座（配对），iii）分析内发生的位点的DNA损伤，ⅳ）评估的等位基因的位置位点相对于核分室（在我们的研究的镇压着丝粒异染色质），及v）评估协会的细胞核蛋白的基因和?…
Discussion
上面详细介绍了在我们的实验室中使用的技术分析的调节V（D）J重组的免疫球蛋白的TCRA / D位点在发展中淋巴细胞30,31。我们相信，这种技术可以适用于不同的核蛋白质，核车厢及基因位点的检测，在不同的细胞类型。协议的修改可能是必要的，而且在这种情况下的主要步骤，集中有以下几种。首先，可以调整长度通透性取决于细胞类型。二初级抗体孵育的长度也可以被调整?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
我们要感谢Skok实验室，特别是苏珊娜·休伊特，成员的讨论和意见。这项工作是由美国国家卫生补助R01GM086852，RC1CA145746（JAS）的支持。 JAS是一个白血病和淋巴瘤协会的学者。 JC是欧文顿研究所的癌症研究所的研究员。 MM是由美国国家科学基金会资助中西医结合研究生教育和研究培训实习（IGERT 0333389 NSF）的支持。
Materials
| Name of Reagent/Material | Company | Catalogue Number | Comments |
| H2O | Fisher | # BP2470 | |
| RNase A | Sigma | # R4642 | |
| dNTP | Sigma | # DNTP100 | |
| Alexa dUTP | Invitrogen | # C11397 to C-11401 | |
| Cy3 or Cy5 dUTP | Fisher | # 45-001-xxx | |
| DNase I | Roche | # 04536282001 | |
| DNA Pol I | Biolabs | # M0209 | |
| 0.025 μm filters | Millipore | # VSWP02500 | |
| Cot-1 DNA 1 mg/ml | Invitrogen | # 18440 | |
| Hybloc DNA 1 mg/ml | Applied Genetics | # MHB | |
| Salmon sperm | Sigma | # D1626 | powder to be resuspended at 10 mg/ml in H2O |
| NaAc (Sodium Acetate, pH 5.2, buffer solution) | Sigma | # S7899 | |
| Ficoll 400 (Mol Biol grade) | Fisher | # 525 | |
| Polyvinylpyrrolidone (Mol Biol grade) | Fisher | # BP431 | |
| Dextran sulfate powder | Sigma | # D8906 | |
| SSPE (Saline-Sodium Phosphate-EDTA) 20x solution | Fisher | # BP1328 | |
| Formamide | Fisher | # BP227 | |
| Coverslips | Fisher | # 12-548-B | |
| Slides | Fisher | # 12-550 | |
| 6-well plates | Fisher | # 0720080 | |
| PBS, 10x | Fisher | # MT-46-013-CM | |
| Poly-L-lysine solution | Sigma | # P8920 | |
| Paraformaldehyde, prills, 95% | Sigma | # 441244 | |
| Triton-X-100, Mol Biol grade | Sigma | # T8787 | |
| BSA (Bovine Serum Albumin) Fraction V | Fisher | # BP 1600 | |
| Normal goat serum | Vector Labs | # S-1000 | |
| Tween-20, Mol Biol grade | Sigma | # P9416 | |
| SSC (Saline Sodium Citrate) 20x solution | Fisher | # BP1325 | |
| ProLong Gold antifade reagent | Invitrogen | # P36930 | |
| DAPI (4′,6-diamidino-2-phenylindole) | Sigma | # D9542 | |
| Best test one coat rubber cement | Art or office supply stores | ||
| Table 1. Specific reagents and small equipment. |
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Diesen Artikel zitieren
Chaumeil, J., Micsinai, M., Skok, J. A. Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization. J. Vis. Exp. (72), e50087, doi:10.3791/50087 (2013).