PAK1 amplified, overexpressing and non-amplified breast cancer cells were grown on cover slips in the presence of cisplatin and/or the PAK inhibitor PF-3758309 and/or the PARP inhibitor rucaparib. Cells were then fixed with 4% paraformaldehyde for 30 min, washed three times with PBS, permeabilized with 0.5% Triton X-100 for 10 min and blocked with 1% albumin in PBS for 30 min at room temperature. Cells were incubated overnight with antibodies specific for -H2AX (pSer139) (Upstate Biotechnology 05-636, clone JBW301), and Rad51 (Santa Cruz Biotechnology sc8349, clone H-92), washed three times with PBS and incubated with Alexa Fluor 488 conjugated secondary antibodies (Life Technologies). The nucleus were counterstained with DAPI (4′,6′-diamidino-2-phenylindole). Confocal analyses were performed with a Nikon TE2000 confocal microscopy system and the number of foci per cell was calculated by dividing the total number of foci in the frame to the number of cells containing foci.
Te2000 confocal microscopy system
Manufactured by Nikon
The Nikon TE2000 is a confocal microscopy system designed for high-resolution imaging. It is a modular system that allows for the integration of various components to meet specific research needs. The core function of the TE2000 is to provide an optical sectioning capability, enabling the capture of detailed, three-dimensional images of samples.
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Lab products found in correlation
3 protocols using te2000 confocal microscopy system
1
Fluorescence Analysis of DNA Damage and Repair
2
Cell Fixation and Confocal Microscopy
3
Cell Fixation and Confocal Microscopy
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cells were fixed in 4% paraformaldehyde at room temperature for 15 min, and processed as described. Confocal analyses were performed with a Nikon TE2000 confocal microscopy system.
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