Cells were seeded and incubated overnight on coverslips and were fixed for 15 min in 4% paraformaldehyde in PBS, permeabilized in 0.5% Triton X-100-PBS for 15 min and blocked in 3% filtered bovine serum albumin (BSA) in PBS. Coverslips with primary antibodies were diluted in blocking solution and incubated overnight at 4 °C. Alexafluor conjugated secondary antibodies were diluted 1:300 and DAPI (diluted 1:500 in blocking buffer, stock 1 mg/ml), in blocking solution and stained for 45 min at 37 oC in humidifier chamber. Slides were washed thrice with 0.05% Tween 20 in PBS and mounted in Prolong Gold. Slides were imaged using GE DeltaVision Deconvolution microscope and analyzed using Image J. Antibodies used for immunofluorescence were: γ-H2ax S139 (05-636; Millipore), p-Histone H3 (#9706; CST), α-Tubulin (T9026), γ-Tubulin (T5192), Acetylated tubulin (T7451; Sigma) and detyrosinated tubulin (ab48389; Abcam).
γh2ax s139
Manufactured by Merck Group
γH2AX (S139) is a laboratory reagent used to detect the presence of DNA double-strand breaks. It is an antibody that specifically recognizes the phosphorylated form of the histone H2AX at serine 139, which is a marker for DNA damage response. This product can be used in various applications, such as immunofluorescence microscopy, flow cytometry, and western blotting, to monitor DNA damage in cells.
Automatically generated - may contain errors
Lab products found in correlation
Ab150081, by Abcam (2 mentions)
Ab150103, by Abcam (2 mentions)
Fv3000 confocal microscope, by Olympus (2 mentions)
γ tubulin, by Merck Group (1 mentions)
Acetylated tubulin, by Merck Group (1 mentions)
Ab48389, by Abcam (1 mentions)
Axio observer microscope, by Zeiss (1 mentions)
Mowiol, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
T9026, by Merck Group (1 mentions)
Gtx 634 420, by GeneTex (1 mentions)
Rpn20ab, by GE Healthcare (1 mentions)
T6557, by Merck Group (1 mentions)
P chk1 s317, by Cell Signaling Technology (1 mentions)
Pchk2 t68, by Cell Signaling Technology (1 mentions)
Fancd2, by Abcam (1 mentions)
Anti γ h2ax s139, by Merck Group (1 mentions)
Hnrnpd, by Merck Group (1 mentions)
α tubulin, by Cell Signaling Technology (1 mentions)
P p53 ser15, by Cell Signaling Technology (1 mentions)
7 protocols using γh2ax s139
1
Immunofluorescence Staining of Cellular Markers
2
Immunofluorescence Analysis of DNA Damage
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Cells were seeded on coverslips, treated, fixed for 20 min with 2% paraformaldehyde/ 2% sucrose and permeabilized for 15 min with 0.1% Triton-X 100. Following 1 h blocking with 2.5% donkey serum in 0.05% PBS/Tween, coverslips were incubated as needed with primary antibodies: γH2AX S139 (1:1500, #05–636-I, Millipore), 53BP1 (1:1500, #sc-22760, Santa Cruz Biotechnology), cyclin A (1:1000, #GTX-634–420, GeneTex) or Phalloidin (1:50, #A12379, Invitrogen). For BrdU staining (1:500, #RPN20AB, GE Healthcare), cells were fixed with ice-cold methanol (40 s) and acetone (20 s), followed by DNA denaturing in 1.5 N HCl for 40 min. For staining of centrosomes (1:1000, #T6557, Sigma-Aldrich) and microtubules (1:1000, #T9026, Sigma-Aldrich), cells were fixed for 10 min with ice-cold methanol, followed by hydration with PBS. Following 1 h of incubation with primary antibodies, cells were washed (3 x/10 min each) with 0.05% PBS/Tween, incubated for 1 h with anti-donkey Alexa 488 or 546 (1:200, Invitrogen), washed, stained with DAPI (#10236276001, Roche) and mounted on slides with Mowiol (Sigma-Aldrich). Slides were analyzed with ×40 or100 x objectives using an Axio Observer microscope (Zeiss).
3
DNA Damage Response Pathway Analysis
4
Indirect Immunofluorescence of CENP-F and γH2AX
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Indirect immunofluorescence was performed as described elsewhere [24 ]. Cells were grown on coverslips for 24 h and treated with 4 Gy γ-irradiation (Gammacell40 Exactor unit) or 4 mM hydroxyurea. Cell nuclei were pre-extracted with nuclear extraction buffer (NEB; 10 mM PIPES (pH 6.8), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA (pH 8.0), 0.5% Triton X-100) for 2 min at RT then fixed with 4% paraformaldehyde (PFA) for 10 min at 4°C. Nuclei were blocked in 5% BSA and 0.3% Triton X-100 in PBS, immunoblotted with a primary antibody (1:500 in dilution buffer; 1% BSA and 0.3% Triton X-100 in PBS), followed by secondary antibody (2 μg/mL in dilution buffer). DNA was counterstained with DAPI. Slides were viewed on an Olympus FV3000 confocal microscope. Primary antibodies: CENP-F (H-260; Santa Cruz sc-22791), γH2AX (S139) (Millipore 05–636). Secondary antibodies: α-Rabbit (Abcam ab150081, Alexa Fluor 488), α-Mouse (Abcam ab150103, Alexa Fluor 647). Nuclear foci quantification was performed using CellProfiler.
5
DNA Damage Response Tracking in HeLa Cells
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HeLa cells, grown on glass coverslips, were fixed with 4% paraformaldehyde and permeabilized with 0.5% triton. Samples were blocked 10 min in 1%BSA at RT and incubated 1 h with anti-BrdU (1:200, 347580, BD Biosciences) or anti-γH2Ax S139 (1:600, 05-636, Millipore) 37°C. After washing, samples were incubated 45 min at 37°C with AlexaFluor 594-conjugated chicken anti-rabbit and 488-conjugated rabbit anti-mouse or 555-conjugated goat anti-mouse IgG (H+L) (Life Technologies), and analyzed with a Zeiss LSM100 confocal microscope.
UVC micro-irradiation was performed as described by Suzuki et al. (23 (link)), then the following antibodies were used for protein detection HNRNPD (1:200, 07-260, Millipore), γH2Ax S139 (1:600, 05-636, Millipore).
UVC micro-irradiation was performed as described by Suzuki et al. (23 (link)), then the following antibodies were used for protein detection HNRNPD (1:200, 07-260, Millipore), γH2Ax S139 (1:600, 05-636, Millipore).
6
Immunofluorescence Analysis of DNA Damage
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Indirect immunofluorescence was performed as described elsewhere (21 ). Cells were grown on coverslips for 24 h and treated with 4 Gy γ-irradiation (Gammacell40 Exactor unit) or 4 mM hydroxyurea. Cell nuclei were pre-extracted with nuclear extraction buffer (NEB; 10 mM PIPES (pH 6.8), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA (pH 8.0), 0.5% Triton X-100) for 2 min at RT then fixed with 4% paraformaldehyde (PFA) for 10 min at 4°C. Nuclei were blocked in 5% BSA and 0.3% Triton X-100 in PBS, immunoblotted with a primary antibody (1:500 in dilution buffer; 1% BSA and 0.3% Triton X-100 in PBS), followed by secondary antibody (2 μg/mL in dilution buffer). DNA was counterstained with DAPI. Slides were viewed on an Olympus FV3000 confocal microscope. Primary antibodies: CENP-F (H-260; Santa Cruz sc-22791), γH2AX (S139) (Millipore 05–636). Secondary antibodies: α-Rabbit (Abcam ab150081, Alexa Fluor 488), α-Mouse (Abcam ab150103, Alexa Fluor 647). Nuclear foci quantification was performed using CellProfiler.
7
Western Blot Analysis of DNA Damage Response
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Western blotting was performed as described previously [16 (link)]. Briefly, cells or tumors were lysed with RIPA buffer supplemented with protease inhibitor (#25,765,800, Sigma) and phosphatase inhibitor (#P5726, Sigma) for 30 min. 30 μg of lysates were separated by SDS-PAGE and transferred to nitrocellulose membrane, blocked with 5% BSA for 1 h at room temperature, then incubated with primary antibody overnight at 4 °C. Primary antibodies include: BRCA2 (#ab27976, Abcam); γ-H2AX S139 (#05–636, Millipore, 1:1000); H2AX (#2595, Cell Signaling, 1:1000); α-Tubulin (#2144, Cell Signaling, 1:1000); Actin (#A5316, Sigma 1:30,000); p-Chk1S345(#2348, Cell Signaling, 1:1000); Chk1 (#2360, Cell Signaling, 1:1000); p-p53Ser15(#82,530, Cell Signaling, 1:1000); p53(#2524, Cell Signaling, 1:1000); p-ATMSer1981(#4526, Cell Signaling, 1:1000); ATM (#2873, Cell Signaling, 1:1000); PD-L1 (#ab213480, Abcam, 1:1000). The next day, membranes were washed with PBST (0.1% Tween-20) three times, incubated with secondary antibody for 2 h at room temperature before developing with ECL substrate (#32,106, Thermo Fisher).
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