Protocol detail

Immunofluorescence Analysis of Cellular Damage

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Analysis for the presence of micronuclei, nuclear bridges, and apoptotic nuclei was performed on fibroblast cells plated on coverslips, cultured for several days, fixed with 4% formaldehyde, and stained with DAPI. Indirect Immunofluorescence (IF) was performed on fibroblast cells, as previously described (Yehezkel et al., 2013 (link)) using mouse antiphosphorylated γ-H2AX (05–636; EMD Millipore). Scoring of γ-H2AX foci was done on a BX50 microscope (Olympus).

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Simon A.J., Lev A., Zhang Y., Weiss B., Rylova A., Eyal E., Kol N., Barel O., Cesarkas K., Soudack M., Greenberg-Kushnir N., Rhodes M., Wiest D.L., Schiby G., Barshack I., Katz S., Pras E., Poran H., Reznik-Wolf H., Ribakovsky E., Simon C., Hazou W., Sidi Y., Lahad A., Katzir H., Sagie S., Aqeilan H.A., Glousker G., Amariglio N., Tzfati Y., Selig S., Rechavi G, & Somech R. (2016). Mutations in STN1 cause Coats plus syndrome and are associated with genomic and telomere defects. The Journal of Experimental Medicine, 213(8), 1429-1440.

Publication 2016

γ-H2AX BX50 microscope

Apoptotic Cells fibroblast Dapi Formaldehyde Indirect immunofluorescence Microscope Mouse Nuclei

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Variable analysis

independent variables

Presence of micronuclei
Presence of nuclear bridges
Presence of apoptotic nuclei

dependent variables

Scoring of γ-H2AX foci

control variables

Fibroblast cells plated on coverslips
Fibroblast cells cultured for several days
Fibroblast cells fixed with 4% formaldehyde
Fibroblast cells stained with DAPI
Use of mouse antiphosphorylated γ-H2AX antibody (05–636; EMD Millipore) for Indirect Immunofluorescence (IF)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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