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Antibody to phospho h2ax

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The Antibody to phospho-H2AX is a laboratory reagent used to detect and quantify the presence of phosphorylated histone H2AX, a marker of DNA double-strand breaks. It is a specific antibody that binds to the phosphorylated form of the H2AX histone protein. The antibody can be used in various experimental techniques, such as immunofluorescence, Western blotting, and flow cytometry, to analyze the cellular response to DNA damage.

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Lab products found in correlation

Goat anti mouse alexa 488, by Thermo Fisher Scientific (2 mentions) 40 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions) Axioskop 2, by Zeiss (1 mentions) Chamber slide, by Thermo Fisher Scientific (1 mentions) Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions) Prolong diamond antifade reagent, by Thermo Fisher Scientific (1 mentions) Anti mouse alexa fluor 488, by Thermo Fisher Scientific (1 mentions) Zen software, by Zeiss (1 mentions) Prolong gold anti fade reagent containing dapi, by Thermo Fisher Scientific (1 mentions) Upright fluorescent microscope, by Zeiss (1 mentions)

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Phospho-H2AX (4 citations)

4 protocols using antibody to phospho h2ax

1

Quantifying DNA Damage Foci by Immunofluorescence

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Cells grown in chamber slides were fixed and permeabilized. They next were incubated with antibody to phospho-H2AX (Millipore) followed by goat-anti-mouse-Alexa488 (Invitrogen) then and mounted with Prolong gold anti-fade reagent containing 40, 6-diamidino-2-phenylindole (DAPI; Invitrogen) to visualize nuclei. Slides were then examined by fluorescence microscopy (Carl Zeiss Axioskop 2, Thornwood, NY). Cells were judged as ‘positive’ for γH2AX foci when they displayed 10 or more discrete dots of brightness.
Liu Z.G., Jiang G., Tang J., Wang H., Feng G., Chen F., Tu Z., Liu G., Zhao Y., Peng M.J., He Z.W., Chen X.Y., Lindsay H., Xia Y.F, & Li X.N. (2016). c-Fos over-expression promotes radioresistance and predicts poor prognosis in malignant glioma. Oncotarget, 7(40), 65946-65956.
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2

Quantifying DNA Damage Foci in Cells

Cited 2 times
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Cells were grown in chamber slides (Thermo Fisher), treated as indicated, fixed in 4% neutral buffered formaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 1% bovine serum albumin in PBS-tween containing 5% goat serum. Slides were incubated with antibody to phospho-H2AX (1:500, Millipore) followed by incubation with goat-anti-mouse Alexa555 (1:750, Invitrogen) and mounted with Prolong gold antifade reagent with DAPI (Invitrogen). Cells were analyzed on a Leica SP5 confocal microscope with ×63 objective or EVOS M5000 Fluorescent Microscope with ×63 objective. Cells with 10 or more γH2AX foci were scored as positive51 (link),63 (link)–65 (link). Data presented are mean ± SEM of 2–3 biological replicates with >50 cells scored per experimental condition as indicated in the figure legend.
Hayman T.J., Baro M., MacNeil T., Phoomak C., Aung T.N., Cui W., Leach K., Iyer R., Challa S., Sandoval-Schaefer T., Burtness B.A., Rimm D.L, & Contessa J.N. (2021). STING enhances cell death through regulation of reactive oxygen species and DNA damage. Nature Communications, 12, 2327.
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3

Quantification of DNA Damage Foci

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To visualize foci, cells grown in chamber slides were fixed with 10% neutral buffered formalin (NBF), permeabilized with 0.1% Triton X-100, and blocked with 1% bovine serum albumin in PBS containing 5% FBS. The slides were incubated with antibody to phospho-H2AX (Millipore) followed by incubation with anti-mouse-AlexaFluor488 (Invitrogen) and mounted with Prolong Diamond antifade reagent containing DAPI (Invitrogen) to visualize nuclei. Images were captured on a Zeiss upright fluorescent microscope equipped with a 63 ×oil immersion lens. Automated image analysis to determine the number of foci per nucleus was performed using ZEN software with the advanced Image Processing and Analysis module (Zeiss). Foci determination was quantified in at least 30 cells per condition. Data presented are the mean ± SEM of 3 independent experiments.
Timme C.R., Rath B.H., O’Neill J.W., Camphausen K, & Tofilon P.J. (2018). The DNA-PK Inhibitor VX-984 Enhances the Radiosensitivity of Glioblastoma Cells Grown In Vitro and as Orthotopic Xenografts. Molecular cancer therapeutics, 17(6), 1207-1216.
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4

Visualization of DNA Damage Foci

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To visualize foci, cells grown in chamber slides were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 1% bovine serum albumin in PBS containing 5% goat serum. The slides were incubated with antibody to phospho-H2AX (Millipore) followed by incubation with goat-anti-mouse-Alexa488 (Invitrogen) and mounted with Prolong gold antifade reagent containing DAPI (Invitrogen) to visualize nuclei. Cells were analyzed on a Zeiss upright fluorescent microscope.
Wahba A., Rath B.H., Bisht K., Camphausen K, & Tofilon P.J. (2016). Polysome profiling links translational control to the radioresponse of glioblastoma stem-like cells. Cancer research, 76(10), 3078-3087.
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