Guinea pig anti-TDP-43 polyclonal antibody was reported previously (15 (link)). Antibody to Stra8 was a kind gift from Dr Michael Griswold, Washington State University. Anti-SYCP3 (Abcam; 15093), anti-γH2AX antibody (Millipore Sigma; 05-636), and anti-Sox9 antibody (Millipore; AB5535) were obtained from commercial sources. anti-γH2AX antibody used for IHC was a mouse monoclonal from Millipore 05-636. Guinea pig anti-SP-10 polyclonal antibody was reported previously (63 (link)).
Anti γh2ax antibody
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom
The Anti-γH2AX antibody is a laboratory tool used for the detection and quantification of phosphorylated H2AX histone, a well-established marker of DNA double-strand breaks. This antibody specifically binds to the γ-H2AX protein, allowing for the visualization and analysis of DNA damage response in cellular samples.
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Lab products found in correlation
Ecl kit, by Thermo Fisher Scientific (8 mentions)
Full length caspase 3, by Cell Signaling Technology (4 mentions)
Cleaved caspase 9, by Cell Signaling Technology (4 mentions)
Caspase 7, by Cell Signaling Technology (3 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488 secondary antibody, by Thermo Fisher Scientific (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
710 confocal microscope, by Zeiss (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
Phosphorylated akt ser473, by Cell Signaling Technology (2 mentions)
Phosphorylated atr ser428, by Cell Signaling Technology (2 mentions)
Phosphorylated chk1 ser345, by Cell Signaling Technology (2 mentions)
Phosphorylated cdc2, by Cell Signaling Technology (2 mentions)
Antimouse alexafluor 594 conjugated igg, by Abcam (2 mentions)
P wee1 ser642, by Cell Signaling Technology (2 mentions)
Anti rad51, by Santa Cruz Biotechnology (2 mentions)
P cdc2 tyr15, by Cell Signaling Technology (2 mentions)
Anti phospho chk1 ser345 antibody, by Cell Signaling Technology (2 mentions)
66 protocols using anti γh2ax antibody
1
Antibody Sources for TDP-43, Stra8, SYCP3, and More
2
Immunohistochemistry and Senescence Analysis of Tissues
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Frozen tissues were sectioned and slides prepared by Histoserv, Inc. (Germantwon, MD, USA). Immunohistochemisty of γH2AX was performed following the instruction of the anti‐rabbit HRP‐DAB Cell & Tissue Staining Kit (R&D systems) with an anti‐γH2AX antibody (1:100; Sigma‐Aldrich). For insulin and glucagon staining, slides were fixed in 4% paraformaldehyde, permeabilized in 0.2% Triton X‐100 in PBS, blocked in a solution containing 5% normal donkey serum, 1% bovine serum albumin, 0.2%, gelatin and 0.2% Triton X‐100 in PBS, hybridized with anti‐insulin and anti‐glucagon antibodies together (1:500; Abcam, Cambridge, MA, USA) for 16 h at 4 °C, washed twice in PBS with 0.2% Triton X‐100 for 30 min and once in PBS for 30 min, hybridized with secondary antibodies (1:200, anti‐mouse Alexa Fluor 488 and anti‐rabbit Alexa Fluor 568; Invitrogen, Carlsbad, CA, USA), and then washed as described above. Finally, the slides were mounted with ProLong Gold Antifade reagent containing DAPI (Invitrogen). The expression of senescence‐associated β‐galactosidase was determined following the instruction of a Senescence Detection Kit (BioVision, San Francisco, CA, USA). Images were visualized and captured under a Nikon Eclipse E600 fluorescence microscope (Nikon Inc., Melville, NY, USA).
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Immunoblotting of DNA Damage Signaling
4
Quantifying DNA Damage Foci by Immunofluorescence
5
Western Blot Analysis of Apoptosis Markers
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Example 9
Cells were dissolved into the Laemmli sample buffer on the plate. The whole cell extracts were sonicated, and the proteins in the lysate were resolved using a 4-12% SDS polyacrylamide gel, and the resolved proteins were blotted onto a nitrocellulose membrane. Antibodies specific to H2A.X, cleaved caspase-3, full-length caspase-3, or cleaved caspase-9 were purchased from Cell Signaling Technology (Danvers, Mass.). The anti-γH2A.X antibody was purchased from Millipore. The membrane was blocked with 5% nonfat dry milk and incubated individually with each of these antibodies dissolved in the blocking buffer. After incubation with peroxidase-conjugated secondary antibodies, the protein of interest was detected using an ECL kit purchased from Thermo Fisher Scientific (Waltham, Mass.).
6
Immunoblotting and Immunofluorescence Antibodies
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Antibodies used for immunoblotting and immunofluorescence were as follows: anti-MCM8 and anti-MCM9 antibodies (raised in rabbits; in-house antibodies), anti-MCM2 antibody (Santa Cruz Biotechnology, sc-9839), anti-MCM5 antibody (Santa Cruz Biotechnology, sc-22780), anti-mAID antibody (MBL, M214-3), anti-RFP antibody (MBL, M204-3), anti-RAD51 antibody (BioAcademia, 70001), anti-γH2AX antibody (Millipore, 05-636), anti-53BP1 antibody (Santa Cruz Biotechnology, sc-22760), anti-Histone H3 (Abcam, ab1791; and Active Motif, 39763), anti-α-tubulin (Sigma, 00020911), and anti-phospho-CHK1 (Ser345) antibody (Cell Signaling, 2341). For detection of MCM8-mCherry2, RFP-Booster ATTO 594 (Chromotek, rba594) was used.
7
DNA Damage Analysis in Silenced HeLa Cells
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For FACS analysis, silenced HeLa cells were washed once with PBS, fixed with ice cold 70% EtOH, and stained with DAPI (1:2500). Anti-γ H2AX antibody (catalog no. 05-636, Millipore) was used for DNA damage studies and analyzed according to Huang and Darzynkiewicz (16 (link)) without cell cycle phase determination. Samples were analyzed using a BD FACS Aria III and FACSDiva software (BD Biosciences).
8
Quantifying DNA Synthesis and Damage in HeLa Cells
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For DNA synthesis studies HeLa cells growing on coverslips in a 6-well plate were treated with relevant drugs (as described in the text) and 15 minutes before fixation 10 µM EdU (Berry & Associates, PY 7562) was added. Cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature. Cells were washed three times with PBS, then permeabilised by treating twice for 10 minutes with PBS-TX (PBS/0.1% Triton X-100) at room temperature. Click reaction mix (0.1 mM 6-carboxyfluorescein TEG-Azide (Berry & Associates, FF 6110), 10 mM Sodium-L-Ascorbate and 2 mM Copper-II-Sulphate) was added to the cells for 30 minutes in the dark. Coverslips were washed three times for 5 minutes with 1% BSA 0.5% Tween in PBS at room temperature in the dark to remove excess copper and azide. Nuclei were stained with DAPI and coverslips were mounted using SlowFade Gold Antifade Reagent (Invitrogen, S36936).
For pSer40/41MCM2 and γ∼H2AX staining cells were treated and fixed as above. Primary anti-pSer40/41MCM2 and anti-γ∼H2AX antibody (Millipore, 05-636) were used together with secondary Alexa Fluor 546 goat anti-rabbit antibody (Life Technologies, A11010) or Alexa Fluor 546 goat anti-mouse antibody (Life Technologies, A11003) respectively.
For pSer40/41MCM2 and γ∼H2AX staining cells were treated and fixed as above. Primary anti-pSer40/41MCM2 and anti-γ∼H2AX antibody (Millipore, 05-636) were used together with secondary Alexa Fluor 546 goat anti-rabbit antibody (Life Technologies, A11010) or Alexa Fluor 546 goat anti-mouse antibody (Life Technologies, A11003) respectively.
9
Western Blot Analysis of Intracellular Protein Levels
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Western blot analysis was performed as previously described [29] (link) and was adopted to determine the intracellular protein levels in response to drug treatment. Briefly, cells were harvested after drug treatment and lysed in electrophoresis sample buffer. Proteins were then electrophoretically separated on a sodium dodecyl sulfate–polyacrylamide gel and transferred onto polyvinylidene difluoride membranes (Amersham Biosciences, GE Healthcare Bio-Sciences Corp, Piscataway, NJ). Protein-conjugated membranes were incubated with primary antibodies overnight at 4°C and then incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibody for 1 hour at room temperature. Western blot signals were visualized by chemiluminescence using SuperSignal West Pico chemiluminescence reagent (Pierce, Rockford, IL). Antibodies against AKT, phospho-AKT, Mre11, and FANCD2 were obtained from Santa Cruz Biotechnology (Dallas, TX), whereas antibodies against Nbs1, phospho-Nbs1 (pNbs1), Rad50, Rad51, and β-actin were from Genetex (San Antonio, TX). Antibodies against caspase-3, caspase-7, and poly(ADP-ribose) polymerase (PARP) and secondary antibodies against rabbit and mouse Ig were purchased from Cell Signaling Technology (Danver, MA). The anti-γH2AX antibody was obtained from Millipore (Billerica, MA).
10
Immunofluorescence Assay for γ-H2AX
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Cells were seeded on coverslips. After indicated treatment, cells were washed with PBS and fixed in 4% paraformaldehyde for 15 min. Cells were permeabilized using 0.25% Triton X-100 solution for 5 min and then blocked in 5% bovine serum albumin for 1 h at room temperature. Coverslips were incubated with anti-γ-H2AX antibody (purchased from Millipore 1:500) overnight at 4 °C. After washed with PBS, the cells were incubated with Alexa Fluor 488 donkey anti-mouse IgG (purchased from Invitrogen, 1:1000) for 1 h. The coverslips were washed with PBS and mounted onto slides using proLong gold antifade reagent with DAPI (Life Technologies, US). Slides were visualized on a microscopy system.
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