5-MethylCytosine Antibody (15H61)

Immunocytochemistry/ Immunofluorescence: 5-MethylCytosine Antibody (15H61) [NBP2-59166]

Immunocytochemistry/Immunofluorescence: 5-MethylCytosine Antibody (C.15200003) [NBP2-59166] - Human osteosarcoma (U2OS) cells were stained with the monoclonal antibody against 5-mC. Cells were fixed with 2.5% PFA in PBS for 30 minutes, permeabilized with 0.5% Triton X-100 for 1 hour and treated with 2N HCl for 1 hour followed by 2 x 5 minutes with 0.1 M borate buffer to depurinate the DNA. After blocking with PBS containing 0.1% Triton X-100 and 1% BSA, the cells were immunofluorescently labelled with the 5-mC antibody diluted 1:500 in blocking solution, followed by a goat anti-mouse antibody conjugated to Alexa488. Figure 3A: cells were immunofluorescently labelled with the 5-mC antibody after incubation of the antibody with 50 uM mCTP (left) or with DAPI (right). Figure 3B: staining of the cells with the 5-mC antibody after incubation of the antibody with 50 uM hmCTP and with DAPI.

Immunocytochemistry/ Immunofluorescence: 5-MethylCytosine Antibody (15H61) [NBP2-59166]

Immunocytochemistry/Immunofluorescence: 5-MethylCytosine Antibody (C.15200003) [NBP2-59166] - HeLa cells were stained with the antibody against 5-mC and with DAPI. Cells were fixed with 4% formaldehyde for 10 minutes and blocked with PBS/Triton X-100 containing 1% BSA. The cells were immunofluorescently labelled with the 5-mC antibody (middle) diluted 1:1000 in blocking solution followed by an anti-mouse antibody conjugated to Alexa594. The left panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.

In-situ Hybridization: 5-MethylCytosine Antibody (15H61) [NBP2-59166]

In-situ Hybridization: 5-MethylCytosine Antibody (C.15200003) [NBP2-59166] - To detect methylated chromosomal regions, FISH was performed on metaphase chromosomes from HeLa cells using the antibody against 5-mC. The cells were blocked in metaphase by treatment with colcemid (0.1 ug/mL) for 1 - 2 hours, fixed overnight at -20C with ethanol/glacial acetic acid and treated with 2N HCl for 30 minutes at room temperature. Subsequently, the cells were blocked with PBS containing 1% BSA and 0.1% Triton X-100 and stained with the 5-mC antibody (left) diluted 1:1,000 in blocking solution, followed by an anti-mouse antibody conjugated to Alexa594. The middle panel shows staining of the chromosomes with DAPI. A merge of the two stainings is shown on the right.