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 488 f ab 2 fragment goat anti rabbit igg
Manufactured by Thermo Fisher Scientific
Alexa Fluor® 488 F(ab′)2 fragment goat anti-rabbit IgG is a secondary antibody conjugate designed for detection and visualization of rabbit primary antibodies in various immunoassays. The F(ab′)2 fragment retains antigen-binding capability while reducing non-specific binding.
Automatically generated - may contain errors
Lab products found in correlation
γh2ax, by Cell Signaling Technology (3 mentions)
Alexa 568 f ab 2 fragment goat anti mouse 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 488 f ab 2 fragment goat anti rabbit 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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