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首页> 美国卫生研究院文献>Biological Procedures Online >A sensitive non-radioactive in situ hybridization method for the detection ofchicken IgG γ-chain mRNA: a technique suitable for detecting ofvariety of mRNAs in tissue sections
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A sensitive non-radioactive in situ hybridization method for the detection ofchicken IgG γ-chain mRNA: a technique suitable for detecting ofvariety of mRNAs in tissue sections

机译：一种灵敏的非放射性原位杂交方法检测鸡IgGγ链mRNA：适用于检测鸡IgG的技术组织切片中的各种mRNA

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摘要

We established a sensitive non-radioactive in situ hybridization (ISH) method for the detection of chicken IgG γ-chain mRNA in paraffin sections. RNA probes were transcribed in vitro from cloned chicken IgG CH1 nucleotide sequences with SP6/T7 RNA polymerases in the presence of DIG-UTP. These probes were used for hybridization and were immunodetected using anti-DIG antibodies conjugated to horseradish peroxidase. The immunoreactive products were visualized with DAB-H2O2. IgG γ-chain mRNA-expressing cells were localized in both the spleen and oviductal tissues. This method demonstrated an excellent sensitivity since the ISH signal was clear and the background was negligible. We found that in the spleen IgG γ-chain mRNA-expressing cells were present mainly in the red pulp, whereas in the oviduct they appeared mainly in the mucosal stroma and not in the mucosal epithelium.
机译：我们建立了灵敏的非放射性原位杂交（ISH）方法来检测石蜡切片中的鸡IgGγ链mRNA。在DIG-UTP存在下，用SP6 / T7 RNA聚合酶从克隆的鸡IgG CH1核苷酸序列中体外转录RNA探针。这些探针用于杂交，并使用与辣根过氧化物酶偶联的抗DIG抗体进行免疫检测。免疫反应产物用DAB-H2O2可视化。 IgGγ链mRNA表达细胞位于脾和输卵管组织中。由于ISH信号清晰且背景可忽略不计，因此该方法具有极好的灵敏度。我们发现，在脾脏IgGγ链mRNA表达细胞中主要存在于红髓中，而在输卵管中，它们主要存在于粘膜基质中而不是在粘膜上皮中。

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