Oocytes or embryos were washed with phosphate-buffered saline (PBS), fixed in 3.7% paraformaldehyde (w/v) prepared in PBS containing 0.1% PVA, and permeabilized with 1% Triton X-100 (v/v) for 1 h at 37°C. The samples were blocked with 1% BSA (w/v) for 1 h, incubated overnight with different antibodies at 4°C in a blocking solution, and washed with 1% BSA. The oocytes or embryos were then incubated overnight with anti-γH2AX (pS139, 1:100; Cell Signaling Technology), anti-ATM (pS1981, 1:100; Cell Signaling Technology), and anti-Histone H3 (acetyl K9, Abcam, Cambridge) antibodies at 4°C overnight. The oocytes or embryos were washed three times with PBS containing 1% BSA and were labeled with FITC/TR-conjugated antibody (1:100) for 1 h at room temperature (FITC/TR is fluorescein isothiocyanate and Texas Red). The oocytes were then counterstained with 5 μg/mL Hoechst 33342 for 15 min, washed three times with PVA–PBS, mounted on a glass slide, and examined using an LSM 710 META confocal laser-scanning microscope (Zeiss, Jena, Germany).
Anti histone h3 acetyl k9
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-histone H3 (acetyl K9) is a primary antibody that recognizes histone H3 acetylated at lysine 9. It is used to detect and quantify acetylation of histone H3 at lysine 9 in various applications such as Western blotting, immunohistochemistry, and chromatin immunoprecipitation (ChIP).
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Lab products found in correlation
Anti histone h4 acetyl k8, by Abcam (4 mentions)
Anti hdac1, by Abcam (2 mentions)
Lsm 710 meta confocal laser scanning microscope, by Zeiss (1 mentions)
Anti atm ps1981, by Cell Signaling Technology (1 mentions)
Fluoview 1000, by Olympus (1 mentions)
Anti igg, by Santa Cruz Biotechnology (1 mentions)
Anti histone h3 trimethyl k27, by Abcam (1 mentions)
Magna chip kit, by Merck Group (1 mentions)
Anti hdac3, by Santa Cruz Biotechnology (1 mentions)
Anti p53 do 1, by Santa Cruz Biotechnology (1 mentions)
Cfx96 real time analyzer, by Bio-Rad (1 mentions)
Quantifast sybr green reagent, by Qiagen (1 mentions)
Protein a g dynabeads, by Thermo Fisher Scientific (1 mentions)
Anti acetyl histone h4, by Merck Group (1 mentions)
Simplechip plus enzymatic chromatin ip kit, by Cell Signaling Technology (1 mentions)
Anti histone h3 acetyl k27, by Abcam (1 mentions)
S220 ultrasonicator, by Covaris (1 mentions)
Anti nf κb p65, by Santa Cruz Biotechnology (1 mentions)
Mini protean 3 system, by Bio-Rad (1 mentions)
Amersham ecl western blotting detection kit, by Cytiva (1 mentions)
14 protocols using anti histone h3 acetyl k9
1
Immunofluorescence Analysis of DNA Damage Response
2
Quantitative Immunocytochemistry of Histone Acetylation
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Immunocytochemistry was performed as described previously.25 (link) Cells were treated once daily for 48 hours with 0–1 mM VPA. Primary antibody incubation (anti-histone H3 (acetyl K9) (rabbit polyclonal; Abcam) and anti-histone H4 (acetyl K8) (rabbit polyclonal; Abcam)) was performed overnight at 4°C. RRX- and FITC-conjugated (Jackson ImmunoResearch Laboratories) were used as secondary antibodies. Omission of primary antibodies resulted in a complete loss of detectable signal.
Imaging was performed with use of a laser-scanning confocal microscope (Olympus Fluoview 1000). Microscope settings were kept constant across the experimental conditions. We quantified the fluorescence signal intensity per cell with ImageJ.
Imaging was performed with use of a laser-scanning confocal microscope (Olympus Fluoview 1000). Microscope settings were kept constant across the experimental conditions. We quantified the fluorescence signal intensity per cell with ImageJ.
3
ChIP-qPCR Assay for Chromatin Immunoprecipitation
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The ChIP assay was performed using Magna ChIP™ Kit (MerckMillipore) following the manufacturer's protocol. ChIP assay was performed as previously described.21 Chromatin from cells was fixed and immunoprecipitated using 2 mg of anti‐ATF6α, anti‐Histone H3 (trimethyl K27) (Abcam), anti‐Histone H3 (acetyl K9) (Abcam), or irrelevant antibody (anti‐IgG, Santa Cruz Biotechnology). The purified and eluted DNA fragments were then quantified by qRT‐PCR assays. The primers are listed in Table S1 .
4
Immunohistochemical Analysis of Histone Modifications
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A senior clinical neuropathologist reviewed histology of each specimen and performed marking on hematoxylin and eosin–stained sections. We reported details on TMA construction and immunohistochemical staining previously.13 (link) The following primary antibodies were used: anti-histone H4 (acetyl K8) (Rabbit polyclonal; Abcam) and anti-histone H3 (acetyl K9) (Rabbit polyclonal; Abcam). The signal was developed with 3,3′-diaminobenzidine. Nuclei were counterstained with hematoxylin.
Researchers were blinded to the clinical data during evaluation of the protein expression. The percentage of nuclear staining and/or cytoplasmatic staining was scored as follows: 0, no positive staining; 1, 1%–25% positive staining; 2, 26%–50% positive staining; 3, 51%–75% positive staining; and 4, 76%–100% positive staining. A mean staining score was computed for each patient.
Researchers were blinded to the clinical data during evaluation of the protein expression. The percentage of nuclear staining and/or cytoplasmatic staining was scored as follows: 0, no positive staining; 1, 1%–25% positive staining; 2, 26%–50% positive staining; 3, 51%–75% positive staining; and 4, 76%–100% positive staining. A mean staining score was computed for each patient.
5
Chromatin Immunoprecipitation (ChIP) Assay
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Chromatin immunoprecipitation (ChIP) assays were performed according to the manufacturer's instructions (Millipore). Each cross-linked sample was immunoprecipitated using anti-Hes1 (Abfrontier), anti-Hes6 (antiserum), anti-HDAC3 (Santa Cruz Biotechnology), or anti-histone H3 (acetyl K9, Abcam, Cambridge, MA, USA) antibodies and the DNA was purified by phenol/chloroform extraction followed by ethanol precipitation. For PCR, the following primers were used for the Hes1 N-box, 5'-TCCTTTTGATTGACGTTGTAGC-3' and 5'-GCACTATTCCAGGACCAAGG-3'.
6
Histone Acetylation Analysis in GBM
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Fresh-frozen GBM tissue from 14 patients (VPA: n = 8, no AED: n = 6) was available for western blotting. Protein extraction was performed with RIPA buffer containing protease and phosphatase inhibitors. Fifty nanograms of protein lysate were used for western blotting, which was performed as described earlier. We used the following primary antibodies: anti-histone H4 (acetyl K8) (rabbit polyclonal; Abcam), anti-histone H3 (acetyl K9) (rabbit polyclonal; Abcam), GAPDH (Rabbit; Sigma-Aldrich).
7
ChIP-qPCR Protocol for Chromatin Immunoprecipitation
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ChIP experiments were performed from 30–50 μg MCF-7 chromatin essentially as previously described (Chapman et al., 2013 (link)). Briefly, chromatin was immunoprecipitated using 3 μg anti-p53 (DO-1, Santa Cruz), 1.5 μg anti-RNAP2 CTD (phospho-S5; Clone 4H8), 2.5μg Anti-Histone H3 (acetyl K9) (ab4441, Abcam), or 2.5 μg anti-acetyl-histone H4 (06-598, Millipore) antibody coupled to 25 μl Protein-A/G Dynabeads (Life Technologies). DNA quantities recovered in control IgG ChIP experiments were consistently below the detectable range. Relative quantities of ChIP-enriched DNA were calculated relative to total input chromatin by qPCR in triplicate on a CFX96 Real-Time Analyzer (Bio-Rad) using Quantifast SYBR Green reagent (QIAGEN) and locus-specific primer pairs (sequences in Supplemental Experimental Procedures ).
8
ChIP Assay for Transcription Factor Binding
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The ChIP assays were performed with a Simple ChIP Plus Enzymatic Chromatin IP kit (Cell Signaling Technology #9005) according to the manufacturer’s recommendations. Immunoprecipitated DNA was used as a template for real time quantitative PCR reactions. Antibodies and oligonucleotides used as primers (Tables 4 and 5
9
Histone Acetylation Profiling by ChIP
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Suspension cells were grown and fixed as described above but without EdU labeling. For each biological replicate, one-half flask of cells was used. Chromatin was extracted and immunoprecipitation performed as previously described (Lee et al., 2010) except that chromatin was sheared using a Covaris S220 Ultrasonicator with the SonoLab7 software and the following parameters: peak incident power 200, duty cycle 10, cycles per burst 200, time per treatment 60 s, number of treatments 3. The antibodies were antihistone H3 acetyl K27 (Abcam, cat no. ab4729), antihistone H3 acetyl K5 (Abcam, cat no. ab51997), and antihistone H3 acetyl K9 (Abcam, cat no. ab 12179).
10
Western Blot Analysis of Protein Targets
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Total cell protein was prepared using RIPA Lysis Buffer (Beyotime Institute of Biotechnology). Protein was measured using a BCA protein Assay Kit (CWBIO). Proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes using a Bio-Rad Mini PROTEAN 3 system (Bio-Rad Laboratories, Inc.). The membranes were blocked with PBS containing 5% milk and 0.1% Tween-20 at room temperature for 1 h. The primary antibodies were as follows: β-actin (mouse monoclonal, dilution 1:10,000; A4551) was from Sigma-Aldrich; Merck KGaA. Lamin A (mouse monoclonal, dilution 1:1,000; sc-71481) was obtained from Santa Cruz Biotechnology, Inc. Anti-NF-κB p65 (rabbit polyclonal, dilution 1:1,000; ab16502), anti-Histone H3 acetyl K9 (rabbit polyclonal, dilution 1:5,000, ab4441), anti-HDAC1(rabbit polyclonal, dilution 1:5,000, ab109411) and anti-IκB-α (rabbit polyclonal, dilution 1:5,000, ab32518) were purchased from Abcam. Horseradish peroxidase-conjugated anti-mouse (1:2,500 dilution) or anti-rabbit (1:2,500 dilution) secondary antibodies were purchased from Bioworld Technology, Inc. Immunoreactive bands were visualized by using the Amersham ECL Western Blotting Detection Kit (Cytiva) according to the manufacturer's instructions. β-actin served as a loading control (19 (link),23 (link)).
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