Department of Molecular Cell Biology, GROW-School for Oncology and Reproduction, Maastricht University Medical Center, P.O. Box 616, 6200 MD, Maastricht, The Netherlands.
Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, Jazan University, Jazan, Kingdom of Saudi Arabia.
Histochem Cell Biol. 2023 Jun;159(6):513-526. doi: 10.1007/s00418-023-02187-4. Epub 2023 Apr 3.
This study compares three different pretreatment protocols for the immunohistochemical detection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in nuclear DNA. The human biological samples analyzed included formalin-fixed and paraffin-embedded (FFPE) normal squamous epithelium, ethanol-fixed cultured cells, and metaphase chromosomes. The antigen retrieval methods included low pH Citrate and high pH Tris-ethylenediaminetetraacetic acid (EDTA) protocols, as well as a method using Pepsin pretreatment combined with HCl for DNA denaturation. A gradual increase in the detection levels of 5-mC and 5-hmC was observed when going from Citrate via Tris/EDTA to Pepsin/HCl retrieval. While the Citrate retrieval protocol was the least efficient for the detection of 5-mC and 5-hmC, it did preserve nuclear morphology and enabled visualization of differences in intra- and internuclear distribution patterns in tissue and cell culture samples by single- and double-fluorescence detection. Quantification of (hydroxy)methylation levels in FFPE material demonstrated a significant heterogeneity and differences in 5-mC and 5-hmC levels within and between nuclei in the different compartments of normal squamous epithelium. It was concluded that immunohistochemical detection of 5-mC and 5-hmC enables the correlation of these DNA modifications with histomorphological features in heterogeneous tissues, but this is influenced by different pretreatment protocols that must be carefully chosen to allow an appropriate interpretation of these epigenetic switches.
本研究比较了三种不同的预处理方案，用于检测核 DNA 中的 5-甲基胞嘧啶（5-mC）和 5-羟甲基胞嘧啶（5-hmC）的免疫组织化学染色。分析的人类生物样本包括福尔马林固定石蜡包埋（FFPE）的正常鳞状上皮、乙醇固定培养细胞和中期染色体。抗原修复方法包括低 pH 柠檬酸盐和高 pH 三羟甲基氨基甲烷-乙二胺四乙酸（EDTA）方案，以及使用胃蛋白酶预处理结合 HCl 进行 DNA 变性的方法。从柠檬酸盐到 Tris/EDTA 再到胃蛋白酶/盐酸，5-mC 和 5-hmC 的检测水平逐渐增加。虽然柠檬酸盐回收方案对 5-mC 和 5-hmC 的检测效率最低，但它确实保留了核形态，并通过单荧光和双荧光检测使组织和细胞培养样本中核内和核间分布模式的差异可视化。对 FFPE 材料中（羟）甲基化水平的定量分析表明，正常鳞状上皮不同隔室中核内和核间的 5-mC 和 5-hmC 水平存在显著异质性和差异。结论是，5-mC 和 5-hmC 的免疫组织化学检测能够将这些 DNA 修饰与异质组织中的组织形态学特征相关联，但这受到不同预处理方案的影响，必须仔细选择这些方案，以允许对这些表观遗传开关进行适当的解释。
