Immunocytochemical Analysis of DNA Damage Markers

Immunocytochemistry was performed as described previously [26 (link)]. Primary antibodies to RAD51 (Millipore, MA, USA) (1:500 dilution) and γH2AX (Millipore, MA, USA) (1:500 dilution) and secondary antibodies to Alexa Fluor 488-conjugated chicken anti-mouse IgG and Alexa Fluor 568-conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, CA) (1:100 dilution) were used for analysis. Nuclei were visualized by staining with DAPI. The slides were briefly counterstained and analyzed by confocal fluorescence microscopy (Carl-Zeiss MicroImaging Inc., Oberkochen, Germany). The number of RAD51- and γH2AX-foci was evaluated in a mean of 100 cells.

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Miyasaka A., Oda K., Ikeda Y., Wada-Hiraike O., Kashiyama T., Enomoto A., Hosoya N., Koso T., Fukuda T., Inaba K., Sone K., Uehara Y., Kurikawa R., Nagasaka K., Matsumoto Y., Arimoto T., Nakagawa S., Kuramoto H., Miyagawa K., Yano T., Kawana K., Osuga Y, & Fujii T. (2014). Anti-tumor activity of olaparib, a poly (ADP-ribose) polymerase (PARP) inhibitor, in cultured endometrial carcinoma cells. BMC Cancer, 14, 179.

Publication 2014

RAD51 γH2AX Alexa Fluor 488-conjugated chicken anti-mouse IgG Alexa Fluor 568-conjugated goat anti-rabbit IgG confocal fluorescence microscopy

Alexa 568 Alexa fluor 488 Anti igg Antibodies Cells Chicken Dapi Dilution Fluorescence microscopy Goat Immunocytochemistry Mouse Nuclei Rabbit

Corresponding Organization :

Other organizations : The University of Tokyo, Teikyo University, Yokohama Welfare Service Association, National Center for Global Health and Medicine

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Variable analysis

independent variables

Primary antibody to RAD51 (1:500 dilution)
Primary antibody to γH2AX (1:500 dilution)
Secondary antibody to Alexa Fluor 488-conjugated chicken anti-mouse IgG (1:100 dilution)
Secondary antibody to Alexa Fluor 568-conjugated goat anti-rabbit IgG (1:100 dilution)

dependent variables

Number of RAD51-foci per cell
Number of γH2AX-foci per cell

control variables

Staining of nuclei with DAPI
Brief counterstaining of slides
Analysis by confocal fluorescence microscopy

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