Immunohistochemical Analysis of Epigenetic Markers

Free-floating sections (40 µm) were incubated in 0.1 M PBS containing 5% normal donkey serum and 0.3% Triton X-100 for 1 h, and subsequently incubated overnight with specific primary antibodies (NeuN: Millipore, Burlington, MA, USA, 5-mc: Active Motif, Carlsbad, CA, USA, Dnmt3a, Dnmt3b, and Dnmt 1: Santa Cruz biotechnology, Inc., Dallas, TX, USA) in 2% normal donkey serum (Vector Laboratories, Burlingame, CA, USA) in PBS at 4 °C and incubated with a 1:200 dilution of Alexa Fluor-conjugated donkey anti-rabbit (488) or donkey anti-mouse (546) antibodies (Invitrogen, Grand Island, NY, USA) for 1 h at room temperature and mounted on glass slides using Vectashield (Vector Laboratories, Burlingame, CA, USA). Fluorescent signals were evaluated on a confocal microscope (LSM 710, Carl Zeiss, Oberkochen, Germany) [55 (link)].