Growing oocytes collected at postnatal day 7 were fixed in 3.7% paraformaldehyde in PBS for 20 min, washed with PBS containing 0.1% BSA, permeabilized with 0.5% Triton X-100 for 15 min. The cells were denatured with 4 N HCl for 10 min, neutralized with 100 mM Tris-HCl (pH 8.5) for 20 min, and then incubated with 1/500 anti-5mC (Eurogentec) and 1/500 anti-5hmC (Active Motif) primary antibodies for 1 h at room temperature. After washing with in PBS with BSA, the cells were incubated with 1/250 fluorescein isothiocyanate-conjugated anti-mouse IgG (Jackson Immuno-Research) and 1/250 rhodamine-conjugated anti-rabbit IgG (Jackson Immuno-Research) for 1 h. The oocytes were then mounted on a glass slide in VECTASHEILD medium with DAPI (Vector Laboratory) and observed under a CSU-10 confocal laser scanning microscope (Yokogawa) with an ImagEM EM-CCD camera (Hamamatsu).
Anti 5mc
Manufactured by Eurogentec
Sourced in Belgium
The Anti-5mC is a laboratory equipment product that detects and measures the presence of 5-methylcytosine (5mC) in DNA samples. It is a tool used in epigenetic research.
Automatically generated - may contain errors
Lab products found in correlation
Anti 5hmc, by Active Motif (3 mentions)
Fluorescein isothiocyanate conjugated anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Rhodamine conjugated anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Imagem emccd camera, by Hamamatsu Photonics (1 mentions)
Ecl western blotting detection reagent, by Thermo Fisher Scientific (1 mentions)
Methylated dna standard kit, by Active Motif (1 mentions)
Protein g magnetic dynabeads, by Thermo Fisher Scientific (1 mentions)
Bio dot apparatus assembly, by Bio-Rad (1 mentions)
4 protocols using anti 5mc
1
Immunohistochemical analysis of 5mC and 5hmC
2
Methylation Analysis of P. falciparum DNA
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South-western blotting assays of P. falciparum genomic DNA and DNA standards (Methylated DNA standard kit from ActiveMotif) were performed as previously described (34 (link)), but without using a DNA-binding protein. DNA prepared as described above from matched volumes of WBC-free uninfected blood served as a control. Primary antibodies used include anti-5mC (rabbit monoclonal; Abnova), anti-5mC (mouse monoclonal; Eurogentec), anti-5hmC (mouse monoclonal; ActiveMotif) and anti-5hmC (mouse monoclonal; Millipore) antibodies. No cross-reactivity was observed between 5hmC and 5mC, 5fc, 5cac and 5C for the anti-5hmC antibodies (Source: Active motif). HRP-conjugated secondary antibodies include anti-rabbit IgG and anti-mouse IgG from GE LifeSciences that were compatible with ECL Western Blotting Detection Reagents (Thermo Fisher Scientific).
3
Methylated and Hydroxymethylated DNA Detection
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Genomic DNA from mouse brains was prepared using a phenol–chloroform method. The methylated DNA immunoprecipitation (MeDIP) and hydroxymethylated DNA immunoprecipitation (hMeDIP) assays were performed as previously described.13 (link),26 (link) Briefly, genomic DNA was denatured and then immunoprecipitated with an anti-5mC (Eurogentec, Liège, Belgium) or an anti-5hmC (Active Motif) antibody and protein G magnetic Dynabeads (Thermo Fisher Scientific). After washing three times, beads were treated with protein K for 4 hours. DNA was prepared with phenol–chloroform and precipitated using cold ethanol, and immunoprecipitated DNA was analyzed by qPCR. All primers are listed in Table S2 .
4
Dot-blot analysis of 5hmC and 5mC
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Genomic DNA was extracted using the Promega purification kit (A1620). DNA samples were denatured in denaturing buffer (0.4 M NaOH/10 mM EDTA) for 10 minutes at 95°C and neutralized with equal volumes of 2 M NH4OAc (pH 7.0). The DNA was then spotted on a nitrocellulose membrane using a Bio-Dot Apparatus Assembly (Bio-Rad). The membrane was air dried, cross-linked by Spectrolinker XL-1000 (120 mJ/cm2), and detected with anti-5hmC (active motif, 1:5000) or anti-5mC (Eurogentec, 1:2500) antibodies. The membrane was stained with methylene blue.
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