A method for combining RNAscope in situ hybridization with immunohistochemistry in thick free-floating brain sections and primary neuronal cultures.
A method for combining RNAscope in situ hybridization with immunohistochemistry in thick free-floating brain sections and primary neuronal cultures.
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DOI：
10.1371/journal.pone.0120120
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Kanaan NM
中科院分区：
文献类型：
--
作者：
Grabinski TM;Kneynsberg A;Manfredsson FP;Kanaan NM
原位杂交（ISH）是一种极其有用的工具，可用于在众多研究领域中将基因表达及表达变化定位到组织样本中的特定细胞群。通常，一个研究小组会付出巨大努力来设计、生成、验证，然后在薄的或超薄的石蜡包埋组织切片中使用原位探针。虽然将ISH和免疫组织化学（IHC）相结合是一种成熟的技术，但在厚的游离组织切片中，将具有单转录本敏感性的商业化ISH检测方法RNAscope ISH与IHC相结合尚未见报道。在此，我们提供了一种在大脑厚的游离组织切片中将RNAscope ISH与IHC相结合的实验方案，该方案能够在单个细胞中同时对基因和蛋白质进行共定位。这种方法在大鼠脑切片中与多种ISH探针（例如富含脯氨酸的小重复序列1a、βIII - 微管蛋白、tau蛋白和β - 肌动蛋白）以及IHC抗体染色（例如酪氨酸羟化酶、βIII - 微管蛋白、NeuN和胶质纤维酸性蛋白）配合良好。此外，我们提供了在原代神经元培养物中结合ISH - IHC双重染色以及在大脑厚的游离组织切片中进行双重ISH标记的实例。最后，我们强调了RNAscope检测被病毒载体转导的神经元中异位DNA的能力。RNAscope ISH是一种商业化技术，它利用一种分支的或“树状”原位方法来实现超灵敏的单转录本检测。免疫组织化学是一种可靠的用于鉴定细胞群中特定蛋白质的方法。将一种灵敏且通用的寡核苷酸检测方法与一种成熟且通用的蛋白质检测方法相结合，是在使用游离组织切片的研究中的一项重大进展。
In situ hybridization (ISH) is an extremely useful tool for localizing gene expression and changes in expression to specific cell populations in tissue samples across numerous research fields. Typically, a research group will put forth significant effort to design, generate, validate and then utilize in situ probes in thin or ultrathin paraffin embedded tissue sections. While combining ISH and IHC is an established technique, the combination of RNAscope ISH, a commercially available ISH assay with single transcript sensitivity, and IHC in thick free-floating tissue sections has not been described. Here, we provide a protocol that combines RNAscope ISH with IHC in thick free-floating tissue sections from the brain and allows simultaneous co-localization of genes and proteins in individual cells. This approach works well with a number of ISH probes (e.g. small proline-rich repeat 1a, βIII-tubulin, tau, and β-actin) and IHC antibody stains (e.g. tyrosine hydroxylase, βIII-tubulin, NeuN, and glial fibrillary acidic protein) in rat brain sections. In addition, we provide examples of combining ISH-IHC dual staining in primary neuron cultures and double-ISH labeling in thick free-floating tissue sections from the brain. Finally, we highlight the ability of RNAscope to detect ectopic DNA in neurons transduced with viral vectors. RNAscope ISH is a commercially available technology that utilizes a branched or “tree” in situ method to obtain ultrasensitive, single transcript detection. Immunohistochemistry is a tried and true method for identifying specific protein in cell populations. The combination of a sensitive and versatile oligonucleotide detection method with an established and versatile protein assay is a significant advancement in studies using free-floating tissue sections.
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影响因子：
14.8
作者：
Kaech, Stefanie;Banker, Gary
通讯作者：
Banker, Gary
影响因子：
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作者：
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Luo, Yuling
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CLEMENTS, JE
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作者：
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作者：
Huebner, Eric A;Strittmatter, Stephen M
通讯作者：
Strittmatter, Stephen M