Detection of Low-Abundance DNA Modifications Using Signal Amplification-Based Immunocytochemistry.
Detection of Low-Abundance DNA Modifications Using Signal Amplification-Based Immunocytochemistry.
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DOI：
10.1007/978-1-0716-0876-0_14
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发表时间：
2021
影响因子：
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通讯作者：
Abdulkadir Abakir;A. Ruzov
中科院分区：
文献类型：
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作者：
Abdulkadir Abakir;A. Ruzov
免疫细胞化学可用于评估表观遗传修饰的空间分布和相对水平。虽然传统的免疫染色已被用于检测动物细胞和组织中的5-甲基胞嘧啶（5 mC）的几十年，基于使用荧光团缀合的二抗的技术的灵敏度并不总是足够的研究DNA修饰是不丰富的DNA相比，5 mC。在这里，我们描述了一种灵敏的免疫细胞化学方案，该方案利用过氧化物酶缀合的第二抗体与催化的报告物沉积偶联，并允许检测低丰度的非规范碱基（例如，哺乳动物DNA中的5-羧基胞嘧啶，5caC，5-甲酰基胞嘧啶，5 fC，5-羟甲基尿嘧啶，5 hmU）。该方法可用于评估DNA修饰的水平和核分布，并允许它们与动物细胞中的蛋白质标记物共定位。
Immunocytochemistry can be instrumental in assessing the spatial distribution and relative levels of epigenetic modifications. Although conventional immunostaining has been utilized for the detection of 5-methylcytosine (5mC) in animal cells and tissues for several decades, the sensitivity of techniques based on the use of fluorophore-conjugated secondary antibodies is not always sufficient for studying DNA modifications that are less abundant in DNA compared with 5mC. Here we describe a protocol for sensitive immunocytochemistry that utilizes peroxidase-conjugated secondary antibodies coupled with catalyzed reporter deposition and allows for detection of low-abundance noncanonical bases (e.g., 5-carboxylcytosine, 5caC, 5-formylcytosine, 5fC, 5-hydroxymethyluracil, 5hmU) in mammalian DNA. This method can be employed for evaluation of the levels and nuclear distribution of DNA modifications and permits their colocalization with protein markers in animal cells.