Kim, Jung-Ae; Kruhlak, Michael; Dotiwala, Farokh; Nussenzweig, Andre; Haber, James E. (2021). CIL:13407, Mus musculus, fibroblast. In Cell Image Library. UC San Diego Library Digital Collections. Dataset. https://doi.org/10.6075/J0J67FMK

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.

• 2021

• Dotiwala, Farokh
• Haber, James E.
• Kim, Jung-Ae
• Kruhlak, Michael
• Nussenzweig, Andre

Preparation: formaldehyde fixed tissue
Relation to intact cell: dispersed cells in vitro
Item type: recorded image; photomultiplier tube (PMT)
Imaging mode: single-spot confocal microscopy; fluorescence microscopy
Parameter imaged: fluorescence emission
Source of contrast: distribution of a specific protein; differences in adsorption or binding of stain
Visualization methods: primary antibody plus labeled secondary antibody; 4',6-diamidino-2-phenylindole (DAPI); Alexa Fluor 546
Processing history: linear methods
Data qualification: Processed;spatialmeasurements;intensitiesquantitation

• Cell Image Library Group ID: 5791

• Mus musculus

• Fibroblast
• Nuclear chromatin
• Nuclear heterochromatin
• Nucleus

• DNA repair

• data
• image

• No linguistic content; Not applicable

Samplenumber: 13407

Doi: https://doi.org/10.6075/J0J67FMK

Source data

• Source Record in the Cell Image Library: https://doi.org/10.7295/W9CIL13407

Reference

• J Cell Biol. 178: 209-218. 2007. https://www.ncbi.nlm.nih.gov/pubmed/?term=17635934

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2022-08-12