A549 Smad4−/− cells (7500/well) were plated into glass chamber slides (BD Biosciences) and allowed to attach overnight. Cells were treated with etoposide (25μM for 4h) then harvested at various time points post treatment. Cells were washed with PBS, fixed with 4% paraformaldehyde (15min at room temperature), washed with PBS, permeabilized with 100% ice-cold methanol, then washed again with PBS. After blocking (5% normal goat serum/1% BSA/0.1% Triton X-100 for 1h at room temperature) cells were stained overnight at 4°C with pH2AX antibody (1:400, Cell Signaling, in 5% NGS/1% BSA), rinsed with PBS-T then incubated with anti-rabbit Alexa Fluor 594 (1:500, Life Technologies) for 1h at room temperature. Cells were then rinsed with PBS-T and mounted using DAPI Fluoromount-G (Southern Biotech, Birmingham, AL). pH2AX immunostaining was quantified by counting at least 100 nuclei per treatment condition; differences were compared by Chi-squared test.
Ph2ax antibody
Manufactured by Cell Signaling Technology
The PH2AX antibody is a laboratory reagent used for the detection of phosphorylated histone H2AX, a marker of DNA double-strand breaks. The antibody specifically recognizes the phosphorylated form of the H2AX protein, which is an important step in the cellular response to DNA damage.
Automatically generated - may contain errors
Lab products found in correlation
Glass chamber slides, by BD (2 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (2 mentions)
Dapi fluoromount g, by Southern Biotech (2 mentions)
P3130, by Merck Group (1 mentions)
Alexa fluor 488 conjugated anti rabbit, by Jackson ImmunoResearch (1 mentions)
5mc antibody, by Abcam (1 mentions)
Alexa fluor 488 conjugated anti mouse, by Jackson ImmunoResearch (1 mentions)
Smz1500, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
3 protocols using ph2ax antibody
1
Quantifying DNA Damage Response in A549 Cells
2
Quantifying DNA Damage Response in A549 Cells
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A549 Smad4−/− cells (7500/well) were plated into glass chamber slides (BD Biosciences) and allowed to attach overnight. Cells were treated with etoposide (25μM for 4h) then harvested at various time points post treatment. Cells were washed with PBS, fixed with 4% paraformaldehyde (15min at room temperature), washed with PBS, permeabilized with 100% ice-cold methanol, then washed again with PBS. After blocking (5% normal goat serum/1% BSA/0.1% Triton X-100 for 1h at room temperature) cells were stained overnight at 4°C with pH2AX antibody (1:400, Cell Signaling, in 5% NGS/1% BSA), rinsed with PBS-T then incubated with anti-rabbit Alexa Fluor 594 (1:500, Life Technologies) for 1h at room temperature. Cells were then rinsed with PBS-T and mounted using DAPI Fluoromount-G (Southern Biotech, Birmingham, AL). pH2AX immunostaining was quantified by counting at least 100 nuclei per treatment condition; differences were compared by Chi-squared test.
3
Epigenetic Modifications in Embryogenesis
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The embryos at the defined time points after fertilization were fixed by 4% polyformaldehyde overnight at 4°C. Then, they were dechorionated manually and dehydrated with methanol. The whole-mount IFs with Dnmt1 antibody (Santa Cruz Biotechnology, catalog no. sc-20701), 5mC antibody (Abcam, catalog no. ab10805), and pH2AX antibody [Cell Signaling Technology, catalog no. 2577S], were done with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen, catalog no. D1306) staining and performed as previously described (20 (link)). The secondary antibodies were Alexa Fluor 488–conjugated anti-rabbit and Alexa Fluor 488–conjugated anti-mouse (Jackson ImmunoResearch; 1:200 dilution). After staining, embryos were deyolked by tweezers and mounted on glass slides in mounting medium (Sigma-Aldrich, catalog no. P3130) at animal polar upturned position. Images were acquired on 710 or 880 META laser scanning confocal microscope and manipulated by ZEN software. Treated or untreated embryos were anesthetized at desired stages with 0.02% tricaine and mounted in 5% methyl cellulose (Sigma-Aldrich, catalog no. M-6385) for observation, and phenotype pictures were taken under Nikon SMZ1500 microscope.
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