Mouse monoclonal anti dnmt1 antibody

Aortic arch samples fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into consecutive 8 μm thick free-floating sections were incubated with 4% bovine serum albumin (BSA) in PBS for 1 h and then reacted with one of the following antibodies: a rabbit polyclonal anti-MCP1 antibody, 1:200 (Abcam, Cambridge, UK); a rabbit polyclonal anti-VEGF antibody, 1:200 (Abcam); a mouse monoclonal anti-DNMT1 antibody, 1:500 (Abcam); a rabbit polyclonal anti-DNMT3A antibody, 1:300 (Abcam); or a rabbit polyclonal anti-DNMT3B antibody, 1:200 (Abcam) at 4 °C overnight. The sections were washed with PBS and reacted with the appropriate biotinylated secondary antibodies, 1:1000 (Cell Signaling Technology, Danvers, MA, USA) in PBS and visualized using an SABC Elite kit (BosterBio Technology., LTD., Wuhan, China). Quantitation of immunoreactive protein was performed on a random five sections and the images were obtained using a microscope (Olympus). The integrated optical density of each was determined using IPP software (Media Cybernetics, Inc., Silver Spring, MD, USA).

The cells were fixed using 4% paraformaldehyde, permeabilized using 0.5% Triton X-100 in PBS, blocked with 20% NGS (Normal Goat Serum) and treated with a mouse monoclonal anti-DNMT1 antibody (1:500, Abcam, Cambridge, MA, USA; ab13537) as well as a rabbit polyclonal anti-E2F1 antibody (1:500, ab6302; Cell Signaling) overnight at 4°C. After washing and incubating in a secondary antibody for 1 hour, the slides were mounted using a medium containing DAPI (Vector Laboratories). The images were collected using a confocal microscope (LSM 700; Carl Zeiss, Oberkochen, Germany). The image analyses were performed using Image-Pro plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).