Jung-Ae Kim, Michael Kruhlak, Farokh Dotiwala, Andre Nussenzweig, James E. Haber (2011) CIL:13407, Mus musculus, fibroblast. CIL. Dataset

Distribution of phosphorylated H2AX occurs preferentially in open chromatin. Wildtype mouse embryo fibroblasts were treated with the histone deacetylase inhibitor trichostatinA 8 hours before being incubated with the radiomimetic drug neocarzinostatin (NCS) for 1hour prior to being fixed and immunolabeled against the phosphorylated form of histone H2AX and co-stained with DAPI. MEFs were grown on glass coverslips and treated with 10 ng/ml NCS for 1 h or with 1 μM TSA for 8 h and were treated with 10 ng/ml NCS for an additional hour before being fixed in 2% freshly prepared PFA in PBS for 5 min at room temperature. Cells were immunolabeled using monoclonal antiphospho-H2AX antibody (clone JBW301; 1:1,000; Upstate Biotechnology) and goat anti–mouse AlexaFluor546 secondary antibody (Invitrogen). The cells were labeled for 30 min in PBS containing 100 nM DAPI, rinsed with PBS, and mounted on glass microscope slides in glycerol-based mounting media containing N-propyl gallate antifade (Sigma-Aldrich). A stack of confocal optical slices was captured through the depth of the nucleus (Z-axis) using a 63x Plan-Apochromat (N.A. 1.4) objective lens, an optical slice thickness of 800nm, a Z-step size of 200nm, and X-Y pixel size of 70nm. Individual optical slices were background subtracted and contrast adjusted only by stretching the histogram in a linear manner consistently for the different fluorescence channels.
Image corresponds to Fig 6B, right panel in Kim et al. J Cell Biol. 178: 209-218. 2007.

磷酸化H2AX的分布优先出现在开放的染色质区域。野生型小鼠胚胎成纤维细胞在用组蛋白去乙酰化酶抑制剂三环己基庚酸（trichostatinA）处理8小时后，于固定和针对磷酸化组蛋白H2AX形式的免疫标记以及与DAPI共染色前1小时与放射模拟药物新卡巴唑（neocarzinostatin，NCS）孵育1小时。成纤维细胞（MEFs）在玻璃载玻片上生长，并用10 ng/ml的NCS处理1小时或用1 μM的TSA处理8小时，并在用10 ng/ml的NCS额外处理1小时后固定于2%新鲜制备的PFA在磷酸盐缓冲液（PBS）中，室温下孵育5分钟。细胞使用单克隆抗磷酸化-H2AX抗体（克隆号JBW301；稀释比1:1,000；Upstate Biotechnology）和山羊抗小鼠AlexaFluor546二抗（Invitrogen）进行免疫标记。细胞在含有100 nM DAPI的磷酸盐缓冲液中标记30分钟，然后用磷酸盐缓冲液冲洗，并在含有N-丙基没食子酸抗褪色剂（Sigma-Aldrich）的甘油基封片介质中封片于玻璃显微镜载玻片上。通过63x Plan-Apochromat（N.A. 1.4）物镜，以800nm的光切片厚度、200nm的Z步长和70nm的X-Y像素大小，捕获了通过核深度（Z轴）的共聚焦光学切片堆栈。单独的光学切片仅通过线性方式拉伸不同荧光通道的直方图进行背景减除和对比度调整。图像对应于Kim等人在《细胞生物学杂志》第178卷第209-218页的图6B右侧面板。2007年。