Next, 1 × 104 cells were seeded in a 24‐well plate containing slides. After different transfection treatments, medium was placed into a constant temperature oven at 37°C with 5% CO2. Cells were exposed to 8‐Gy radiation. After 24 hours, medium was washed three times with pre‐warmed 1× PBS for 10 minutes each. Air‐dried slides were fixed with 4% formaldehyde in TBS for 15‐20 minutes at room temperature and washed three times with 1× PBS for 10 minutes each. Cells were then permeabilized with 0.2% Triton X‐100 for 4 minutes and washed three times with 1× PBS for 10 minutes each. These cells were then blocked in 5% BSA at room temperature for 30 minutes. γ‐H2AX primary antibody (Abcam) was added at 4°C overnight. Cells were washed three times with 1× PBS for 10 minutes each. Next, Cy3 fluorescently labeled secondary antibody (Abcam) was added for 1 hour at room temperature in darkness. Cells were then washed as before. After soaking in Hoechst's solvent for 15 minutes, the plate was sealed with 95% glycerol. Finally, cells were observed under a fluorescence microscope for photographing, at randomly selected multiple fields of view (800) for the counting of foci. Each experiment was repeated three times.
γ h2ax primary antibody
Manufactured by Abcam
Sourced in United States
The γ‐H2AX primary antibody is a laboratory reagent used in the detection and quantification of phosphorylated histone H2AX, a marker for DNA double-strand breaks. This antibody specifically recognizes the phosphorylated form of histone H2AX, which is a key event in the cellular response to DNA damage.
Automatically generated - may contain errors
Lab products found in correlation
Cy3 fluorescently labeled secondary antibody, by Abcam (1 mentions)
Normal serum, by Vector Laboratories (1 mentions)
Dako lsab 2 system hrp, by Agilent Technologies (1 mentions)
Sytox blue, by Thermo Fisher Scientific (1 mentions)
Click it edu alexa fluor 488 flow cytometry assay kit, by Thermo Fisher Scientific (1 mentions)
Fluorescence conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ma900 multi application cell sorter, by Sony (1 mentions)
4 protocols using γ h2ax primary antibody
1
Radiation-induced DNA Damage Assay
2
Quantifying DNA Damage with γH2AX Foci
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For this, γH2AX foci, a marker for DNA double-strand breaks, was detected via immunofluorescence assay. Post-2 Gy radiation, transfection of A549 cells with siRNA against MMP10 was conducted. At specified times, cells were fixed with chilled methanol/acetone (1 : 1); then, BSA (3%; in PBS) was used for blocking at room temperature for 60 min. Then, cells allowed to bind to a γH2AX primary antibody (1 : 300; Abcam, US) were reacted with the secondary antibody (1 : 1000). Then, confocal and conventional microscopy was used to monitor immunofluorescence; each group recorded the number of γH2AX foci in 30 cells and took the average.
3
Spleen Histology and Immunohistochemistry Analysis
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The spleen tissues were dissected and fixed in 10% neutral formalin and embedded in paraffin. Histological analysis was performed on paraffin-embedded sections stained with hematoxylin and eosin (H&E) using the conventional method. For immunohistochemistry, we performed general methods. Briefly, paraffin tissue sections were deparaffinized and rehydrated with xylene and serial dilutions of ethanol. Then, tissue rehydration was followed by antigen retrieval through boiling in 1 mM citric acid buffer (pH 6.0). Tissue sections were incubated in 3% H2O2 and blocked with 10% normal serum (Vector Labs, Burlingame, CA, USA) and incubated with γ-H2AX primary antibody (Abcam, Cambridge, MA, USA) overnight at 4 °C. Secondary antibody incubations were carried out using Dako LSAB2 System-HRP (Dako, Santa Clara, CA, USA). Tissue sections were visualized with a DAB system (Vector Labs) and counterstaining was performed by H&E. Images were captured using an Aperio ImageScope instrument (Leica Biosystems Inc., Buffalo Grove, IL, USA).
4
Measuring DNA Damage and Repair in ES Cells
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Chd1fl/Δ and Chd1Δ/Δ (established) ES cells were used. Overnight cultured cells were incubated with 10 μM 5-ethynyl-2′-deoxyuridine (EdU) for 1 h. Cells were harvested, fixed in 4% PFA for 15 min, and permeabilized in 0.25% Triton-X 100 in PBS for 5 min on ice. Cells were washed with 1% BSA in PBS and incubated in γH2A.X primary antibody (Abcam, ab2893) for 20 min at room temperature. Following a second wash, cells were incubated with fluorescence-conjugated secondary antibody (Life Technologies) for 20 min at room temperature. Subsequent EdU labeling was conducted according to manufacturer instructions of the Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit ((Life Technologies). SYTOX Blue (Invitrogen) was used to detect DNA content at 1:1000 dilution. Data was collected on a Sony MA900 Multi-Application Cell Sorter, analyzed using FCS Express 7, and plotted using Prism 7.
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