Slides were retrieved and air dried prior to staining. After rinsing in 1x Tris buffered saline (TBS), they were blocked with 1x TBS / 1% bovine serum albumin/10% FBS/0.3 M glycine for 1 hr at room temperature. Slides were then stained with anti-phospho-histone γH2AX (Merck), anti-53bp1 (Bethyl Laboratories) and anti-Insulin (Dako) overnight at +4 °C. Next, the slides were rinsed and incubated with secondary antibodies, anti-rabbit Alexa 488, anti-rabbit Alexa 647, anti-mouse Alexa 488, anti-guinea pig Alexa 547 (Life Technologies) and DAPI for 1 hour at room temperature. Finally, slides were rinsed in 1x TBS before being mounted with Hardset Antifade mounting medium (Vectashield) and a glass coverslip.
Anti phospho histone γh2ax
Manufactured by Merck Group
Sourced in United States, United Kingdom, France
The Anti-phospho-Histone γH2AX is a laboratory reagent used to detect DNA double-strand breaks. It is an antibody that specifically binds to the phosphorylated form of the histone variant H2AX, which is a marker of DNA damage response.
Automatically generated - may contain errors
Lab products found in correlation
Anti beclin 1, by Novus Biologicals (2 mentions)
Anti tpx2, by Santa Cruz Biotechnology (2 mentions)
Anti golph3, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Anti 53bp1, by Fortis Life Sciences (1 mentions)
Anti insulin, by Agilent Technologies (1 mentions)
Hardset antifade mounting medium, by Vector Laboratories (1 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
A9044, by Merck Group (1 mentions)
P0448, by Agilent Technologies (1 mentions)
Anti bax, by Cell Signaling Technology (1 mentions)
Anti bcl 2, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Non reducing laemmli sample buffer, by Bio-Rad (1 mentions)
Amershan imager 600, by GE Healthcare (1 mentions)
Supersignal west femto, by Thermo Fisher Scientific (1 mentions)
T5168, by Merck Group (1 mentions)
Anti tubulin, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
7 protocols using anti phospho histone γh2ax
1
Multicolor Immunofluorescence Staining
2
Western Blot Analysis of hiPSC-CMs
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hiPSC-CMs were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors (all from Sigma-Aldrich). Equal amounts of samples were mixed with non-reducing Laemmli sample buffer (BioRad, Hercules, CA, USA) and denatured at 96 °C for 5 min. Proteins were then separated on 10–12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore). Membranes were blocked with 5% non-fat dry milk powder in Tris-buffered saline. Primary antibodies used for western blotting were as follows: anti-tubulin (1/1000; T5168, Sigma-Aldrich), anti-caspase-3 (1/500; #9662, Cell Signaling Technology, Danvers, MA, USA), anti-cleaved caspase-3 (1/200; #9661, Cell Signaling Technology), anti-Bax (1/500; #5023, Cell Signaling Technology), anti-Bcl-2 (1/200; #4223, Cell Signaling Technology) and anti-phospho-Histone γ-H2AX (1/1000; 05-636-200, Merck Millipore). Secondary antibodies were anti-IgG rabbit (1/5000; P0448 Dako, Santa Clara, CA, USA) and anti-IgG mouse (1/5000; A9044, Sigma-Aldrich). Detection was carried out using peroxidase-conjugated antibodies and SuperSignalTM West Femto (ThermoFisher Scientific). Reactions were visualized using an Amershan Imager 600 (GE Healthcare, Chicago, IL, USA) and quantified with the ImageJ software (ver. 1.53t, NIH, Bethesda, MD, USA).
3
Protein Expression Analysis by Western Blotting
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Cells were lysed in Staph-A buffer (1.6 mM NaH2PO4; 8.6 mM Na2HPO4; 1% Triton X-100; 0.1% SDS; 0.1% NaCl; 0.5% NaDoc; 2 mM AEBSF; 20 mg/mL each of aprotinin and leupeptin) and the obtained protein lysates (100 µg each) were subjected to SDS-PAGE electrophoresis and transferred to PVDF membrane (Millipore, Burlington, MA, USA). Blots were probed with the following monoclonal antibodies: anti-PARP (Abcam, Cambridge, MA, UK), anti-phospho-Histone γH2AX (Millipore), anti-TPX2 (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-c-MYC (Santa Cruz Biotechnology); and polyclonal antibodies: anti-GOLPH3 (Abcam), anti-Beclin-1 (Novus Biologicals, Littleton, CO, USA), anti-LC3A (Cell Signaling Technology Inc., Danvers, MA, USA), anti-MYO18A (Abnova, Taipei, Taiwan), anti-Histone H2AX (Sigma, St Louis, MO, USA), and anti-MYCN (Novus Biologicals). Proteins were visualized by West Dura Extended chemiluminescent detection (Thermo Scientific, Carlsbad, CA, USA) using HRP-conjugated secondary antibodies (Thermo Scientific). Blots were re-probed with anti-β-actin (Santa Cruz Biotechnology) as loading control. Bands signal intensity was measured by densitometry using Image Lab 6.0 software (ChemiDoc, Bio-Rad, Hercules, CA, USA), and normalized to the loading control.
4
Immunofluorescence Quantification of DNA Damage
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After irradiation, the cells on the TW insert membrane (#3460, Corning) were fixed with 4% paraformaldehyde for 15 min, washed with PBS, permeabilized (100 mM TrisCl pH 7.4, 50 mM EDTA, 0.5% Triton100 in H2O) and blocked (3% BSA, 0.1% Tween20, 4xSSC, 7.7 mM NaN3 in H2O). The cells on the membranes were incubated overnight at 4 °C with the primary antibodies anti-phospho histone γH2AX (Millipore, 05-636) and anti-53BP1 (Abcam, ab21083) in PBS. The cells were washed with PBS and incubated with a secondary antibody labeled with Alexa488 (A 2102, Invitrogen) or Cy3 (BA 1034-05, Boster) and DAPI 1 mg/ml (Thermo Scientific) for 90 min at room temperature. The membranes were cut manually and transferred onto slides, mounted with immu-mount and cover-slipped. The γH2AX and 53PB1 foci per nucleus were identified by eye and calculated manually using a Leica DMi8 fluorescent microscope (USA).
5
Immunofluorescence Analysis of Neuroblastoma
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Immunofluorescence analysis was performed on formalin-fixed, paraffin-embedded NB specimens (4 μm-thick) or on cytospins as previously described [90 (link)]. The following antibodies were used: monoclonal anti-phospho-Histone γH2AX (1:20, Millipore) and anti-TPX2 (1:20, Santa Cruz Biotechnology); polyclonal anti-GOLPH3 (1:10, Abcam), and anti-Beclin-1 (1:30 Novus Biologicals). We used isotype matched non-binding mAbs in all antibody staining experiments to avoid nonspecific reactivity. Results were photographically documented using fluorescence microscope Axio Imager M2 equipped with ApoTome System (Carl Zeiss, Oberkoche, Germany). For cytospins, values represent percentages from at least 1000 counted positive and negative cells. For NB specimens, each tumor area tested contained malignant cells as assessed by histologic examination. Quantification of immunofluorescence positive tumor cells was performed on serial tissue sections, thus allowing quantification in tumor areas selected by the pathologist. Tumor cells were identified in each sample using the NB specific marker NCAM (NB56) [91 (link)]. The proportion of immunofluorescence positive cells counted was at least 100 to 1000 cells and reported in the percentage for the subsequent statistical analysis.
6
Apoptosis, DNA Damage, and ROS Detection
7
Quantifying DNA Damage and Autophagy
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Cells were grown on glass coverslip for 24 h. After irradiation for 4, 8, 12, and 24 h, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 for 30 min at room temperature, and stained with antiphospho-histone γ-H2AX (Millipore, France, diluted 1: 250) and the rabbit anti-human light chain 3 antibody (LC3) (1: 200, Abcam, UK) at 4 °C overnight. After washing in PBS, a secondary antibody (1: 100) conjugated to FITC was added and incubated for 1 h at room temperature. Then, cells were incubated with 0.25 mg/mL DAPI for 3 mins. The images were examined under a laser scanning confocal microscope.
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