An optimized protocol for high-throughput in situ hybridization of zebra finch brain.
An optimized protocol for high-throughput in situ hybridization of zebra finch brain.
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DOI：
10.1101/pdb.prot084582
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发表时间：
2014-10-23
影响因子：
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通讯作者：
Mello CV
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文献类型：
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作者：
Carleton JB;Lovell PV;McHugh A;Marzulla T;Horback KL;Mello CV
原位杂交（ISH）是一种灵敏的技术，用于记录mRNA的组织分布。基于地高辛（DIG）标记的探针的显色检测的非放射性方法的进展与乳液放射自显影相比具有增加的空间分辨率，并且当与高分辨率数字成像配对时，允许在组织学背景内以细胞分辨率进行大规模分子谱分析（例如，艾伦脑图谱，GenePaint. org。技术挑战限制了可以在小型实验室环境中研究的基因数量。该方案描述了一种低成本、小足迹、高通量的ISH程序，用于10 μm切片，以记录斑胸草雀脑基因表达（www.zebrafinchatlas.org）。它使用DIG标记的核糖核酸探针合成的cDNA模板可通过鸣禽神经基因组学协会，是基于先前描述的协议，我们已经适应DIG标记的放射性标记的核糖核酸探针。现在已经进一步优化了条件，以产生接近免疫组织化学方法的分辨率、低背景和与高分辨率数字成像兼容的细胞标记。该方案允许技术人员每周处理约180个载玻片，并且可以缩放以适应可以获得冷冻切片的广泛组织。
In situ hybridization (ISH) is a sensitive technique for documenting the tissue distribution of mRNAs. Advances in non-radioactive methods based on chromogenic detection of digoxigenin (DIG)-labeled probes have increased spatial resolution compared to emulsion autoradiography, and when paired with high-resolution digital imaging have allowed for the large scale molecular profiling at cellular resolution within a histological context (e.g. Allen Brain Atlas, GenePaint.org. However, technical challenges restrict the number of genes that can be investigated in a small laboratory setting. This protocol describes a low cost, small footprint, high-throughput ISH procedure for 10 μm sections developed to document brain gene expression in zebra finches (http://www.zebrafinchatlas.org). It uses DIG-labeled riboprobes synthesized from cDNA templates available through the Songbird Neurogenomics Consortium and is based on previously described protocols for radiolabeled riboprobes that we have adapted for DIG-labeled. Conditions have now been further optimized to produce cellular labeling approaching the resolution of immunohistochemical methods, low background, and compatibility with high-resolution digital imaging. This protocol allows a technician to process ~180 slides per week, and can be scaled to accommodate a broad range of tissues for which cryosections can be obtained.