DSB repair foci were analyzed using co-staining of γH2AX and 53BP1, as described previously (32 (link)). Cells were fixed and stained at 0, 4, and 24 h after irradiation with a primary antibody solution as follows: mouse monoclonal anti-phospho-S139-H2AX antibody (1:500, clone JBW301, Millipore, Darmstadt, Germany) and rabbit polyclonal 53BP1 antibody (1:500, Novus Biologicals, Wiesbaden, Germany). Secondary antibody solutions were mouse Alexa-Fluor 594 (1:1,000) and rabbit Alexa-Fluor 488 (1:1,000, both Invitrogen, Karlsruhe, Germany). Cells were mounted in ProLong Gold Antifade Reagent with DAPI (Invitrogen, Karlsruhe, Germany). Immunofluorescence was analyzed using the Leica DM5500 wide-field microscope and LAS-AF software (Leica, Wetzlar, Germany). All experiments were performed at least twice and with 100 counted nuclei per experiment.
Mouse monoclonal anti phospho s139 h2ax antibody
Manufactured by Merck Group
Sourced in Germany
The Mouse monoclonal anti-phospho-S139-H2AX antibody is a laboratory reagent designed for the detection of phosphorylated serine 139 of the H2AX histone protein. This antibody can be utilized in various research applications that involve the analysis of DNA damage response and repair processes.
Automatically generated - may contain errors
Lab products found in correlation
Apotome, by Zeiss (2 mentions)
Mouse monoclonal anti rad51 antibody, by Abcam (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)
53bp1 antibody, by Novus Biologicals (1 mentions)
Mouse alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Rabbit alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Las af software, by Leica camera (1 mentions)
Dm5500 widefield microscope, by Leica camera (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Axio observer, by Zeiss (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
3 protocols using mouse monoclonal anti phospho s139 h2ax antibody
1
Quantification of DNA Double-Strand Break Repair
2
Immunofluorescence Staining of DNA Damage
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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.
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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