Cells grown on cover slips were washed once with cold PBS and fixed with 4% para-formaldehyde/PBS for 10 min. Fixed cells were permeabilised with 0.2% Triton X-100/PBS on ice for 5 min. The cells were incubated overnight with primary antibodies: mouse monoclonal anti-phospho-S139-H2AX antibody (Millipore) at a dilution of 1:300, mouse monoclonal anti-RAD51 antibody (Abcam 14B4) at a dilution of 1:1000. After being washed three times with cold PBS, the cells were incubated for 1 h with secondary anti-mouse Alexa-fluor594 (Invitrogen) at a dilution of 1:500 or anti-rabbit Alexa-fluor488 (Invitrogen) at a dilution of 1:600. The nuclei were counterstained with 4′-6-diamidino-2-phenylindole (DAPI, 10ng/ml). Slides were mounted in Vectashield mounting medium (Vector Laboratories). Immunofluorescence was observed with the Zeiss AxioObserver.Z1 microscope (objectives: ECPlnN 40x/0.75 DICII, resolution 0.44 μm; Pln Apo 63x/1.4Oil DICII, resolution 0.24 μm; EC PlnN 100x/1.3 Oil DICII, resolution0.26 μm and filters: Zeiss 43, Zeiss 38, Zeiss 49). Semi-confocal images were obtained using the Zeiss Apotome, Zeiss AxioCamMRm and Zeiss AxioVision Software.
Mouse monoclonal anti rad51 antibody
Manufactured by Abcam
Sourced in Germany
Mouse monoclonal anti-RAD51 antibody recognizes the RAD51 protein, a key player in homologous recombination and DNA repair.
Automatically generated - may contain errors
Lab products found in correlation
Mouse monoclonal anti phospho s139 h2ax antibody, by Merck Group (2 mentions)
Apotome, by Zeiss (2 mentions)
Alexa fluor 488 anti rabbit, by Thermo Fisher Scientific (2 mentions)
Axiocam mrm, by Zeiss (2 mentions)
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (2 mentions)
Axio observer, by Zeiss (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Xcellence software, by Olympus (1 mentions)
Anti γh2ax, by Merck Group (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
3 protocols using mouse monoclonal anti rad51 antibody
1
Immunofluorescence Staining of DNA Damage
2
Immunofluorescence Assay for DNA Damage and Repair
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Immunofluorescence analysis was performed as previously described [34 (link)]. Briefly, cells grown and treated on glass coverslips were washed with PBS and fixed with 4% para-formaldehyde for 10 min. Fixed cells were permeabilized with 0.2% Triton X-100 on ice for 5 min. The cells were incubated with primary antibodies: anti-γ-H2AX (Millipore, Darmstadt, Germany), mouse monoclonal anti-RAD51 antibody (1:1000, clone 14b4, Abcam, Cambridge, UK), and anti-CenpF (Lifespan Biosciences, Seattle, WA, USA); and secondary antibodies: anti-mouse AlexaFluor594 or anti-rabbit Alexa Fluor-488 (Invitrogen, Karlsruhe, Germany). Nuclei were counterstained with 4′,6-diamidin-2-phenylindol (DAPI). Cells were viewed with an IX81 microscope using a 60× Neofluor objective and Xcellence Software (Olympus, Hamburg, Germany).
γH2AX foci were scored visually in CenpF-negative (G1-phase) and CenpF-positive cells (G2-phase). Generally, non-irradiated cells showed less than five γH2AX foci per cell nucleus. Therefore, data of irradiated cells are presented as the percentage of cells with ≥5 γ-H2AX foci. A minimum of 100 cells in three independent experiments was counted and data are shown as mean values ± standard error of the mean (SEM).
γH2AX foci were scored visually in CenpF-negative (G1-phase) and CenpF-positive cells (G2-phase). Generally, non-irradiated cells showed less than five γH2AX foci per cell nucleus. Therefore, data of irradiated cells are presented as the percentage of cells with ≥5 γ-H2AX foci. A minimum of 100 cells in three independent experiments was counted and data are shown as mean values ± standard error of the mean (SEM).
3
Immunofluorescence Analysis of DNA Damage Markers
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Cells grown on cover slips were washed once with cold phosphate buffered saline (PBS) and fixed with 4% para-formaldehyde/PBS for 10 min. Fixed cells were permeabilized with 0.2% Triton X-100/PBS on ice for 5 min. The cells were incubated overnight with primary antibodies: mouse monoclonal anti-phospho-S139-H2AX antibody (Millipore) at a dilution of 1:300, mouse monoclonal anti-RAD51 antibody (Abcam 14B4) at a dilution of 1:1000, mouse monoclonal anti-RPA antibody (Santa Cruz Biotechnology) at a dilution of 1:600 and rabbit monoclonal anti-CenpF antibody (Lifespan Biosciences) at a dilution of 1:750. After being washed three times with cold PBS, the cells were incubated for 1 h with secondary anti-mouse Alexa-fluor594 (Invitrogen) at a dilution of 1:500 or anti-rabbit Alexa-fluor488 (Invitrogen) at a dilution of 1:600. The nuclei were counterstained with 4′-6-diamidino-2-phenylindole (DAPI, 10 ng/ml). Slides were mounted in Vectashield mounting medium (Vector Laboratories). Immunofluorescence was observed with the Zeiss AxioObserver.Z1 microscope (objectives: ECPlnN 40x/0.75 DICII, resolution 0.44 μm; Pln Apo 63x/1.4Oil DICII, resolution 0.24 μm; EC PlnN 100x/1.3 Oil DICII, resolution 0.26 μm and filters: Zeiss 43, Zeiss 38, Zeiss 49). Semi-confocal images were obtained using the Zeiss Apotome, Zeiss AxioCamMRm and Zeiss AxioVision Software.
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