Cells were seeded onto UV-irradiated coverslips and were treated for 24h with HER2–205, MIX24, or a mock transfection. Cells were processed 24h post-transfection, fixed with 4% formaldehyde and then incubated with ice-cold 100% methanol for 20 minutes followed by a methanol and acetone solution (1:1) for 20 minutes each at −20°C. After washing with PBS, cells were blocked with blocking buffer (4% BSA, 0.2% Triton X-100 in PBS) for 30 minutes and then incubated overnight with the following primary antibodies: γH2AX (1:500; Cell Signaling or Millipore) and 53BP1 (1:100; Santa Cruz) in blocking buffer at 4°C. After three washes, cells were incubated with secondary antibodies Alexa 488 F(ab′)2 fragment goat anti-rabbit IgG or Alexa 568 F(ab′)2 fragment goat anti-mouse IgG (1:1000; Molecular Probes) for 1h at room temperature. Cells were mounted on microscope glass slides with anti-fade mounting media containing DAPI (Life Technologies), and pictures were taken with a Leica SP5 microscope. Immunofluorescence experiments were repeated for validation.
Alexa 568 f ab 2 fragment goat anti mouse igg
Manufactured by Thermo Fisher Scientific
Alexa Fluor 568 F(ab')2 fragment of goat anti-mouse IgG is a secondary antibody conjugate designed for use in immunoassays and other applications requiring the detection of mouse immunoglobulins. The F(ab')2 fragment is generated by pepsin digestion of the full antibody, retaining the antigen-binding regions while removing the Fc portion. The Alexa Fluor 568 dye provides a bright, photostable fluorescent label for visualization and quantification.
Automatically generated - may contain errors
Lab products found in correlation
γh2ax, by Cell Signaling Technology (3 mentions)
Alexa 488 f ab 2 fragment goat anti rabbit igg, by Thermo Fisher Scientific (3 mentions)
Sp5 microscope, by Leica (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Prpa32, by Fortis Life Sciences (1 mentions)
Rad51, by Santa Cruz Biotechnology (1 mentions)
3 protocols using alexa 568 f ab 2 fragment goat anti mouse igg
1
Immunofluorescence Assay for DNA Damage
2
Immunofluorescence Assay for DNA Damage
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Cells were seeded onto UV-irradiated coverslips and were treated for 24h with HER2–205, MIX24, or a mock transfection. Cells were processed 24h post-transfection, fixed with 4% formaldehyde and then incubated with ice-cold 100% methanol for 20 minutes followed by a methanol and acetone solution (1:1) for 20 minutes each at −20°C. After washing with PBS, cells were blocked with blocking buffer (4% BSA, 0.2% Triton X-100 in PBS) for 30 minutes and then incubated overnight with the following primary antibodies: γH2AX (1:500; Cell Signaling or Millipore) and 53BP1 (1:100; Santa Cruz) in blocking buffer at 4°C. After three washes, cells were incubated with secondary antibodies Alexa 488 F(ab′)2 fragment goat anti-rabbit IgG or Alexa 568 F(ab′)2 fragment goat anti-mouse IgG (1:1000; Molecular Probes) for 1h at room temperature. Cells were mounted on microscope glass slides with anti-fade mounting media containing DAPI (Life Technologies), and pictures were taken with a Leica SP5 microscope. Immunofluorescence experiments were repeated for validation.
3
Immunofluorescence Analysis of DNA Damage Response
Check if the same lab product or an alternative is used in the 5 most similar protocols
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MEFs were seeded onto UV-irradiated coverslips and were treated for 24 h with either AG30, MIX30 or a mock transfection. Cells were processed 24 h post-transfection, fixed with 4% formaldehyde and then incubated with ice-cold 100% methanol followed by a methanol and acetic acid solution (1:1) for 20 min at −20°C. After washing with phosphate buffered saline (PBS), cells were blocked with blocking buffer (4% BSA, 0.2% Triton X-100 in PBS) for 30 min and then incubated overnight with the following primary antibodies: γH2AX (1:500; Cell Signaling), 53BP1 (1:100; Santa Cruz), RAD51 (Santa Cruz) and pRPA32 (Bethyl Laboratories) in blocking buffer at 4°C. After three washes, cells were incubated with secondary antibodies Alexa 488 F(ab′)2 fragment goat anti-rabbit IgG or Alexa 568 F(ab′)2 fragment goat anti-mouse IgG (1:1000; Molecular Probes) for 1 h at room temperature. Cells were then mounted on microscope glass slides with anti-fade mounting media containing DAPI (Life Technologies), and pictures were taken with a Leica SP5 microscope. pRPA32 foci were detected using the protocol described by Mirzoeva and Petrini (27 (link)).
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