Anti dnmt1

Sample fixation was performed with a solution of 4% of paraformaldehyde in 0.1 M of PBS (Lonza) [35 (link), 36 (link)]. The following steps were performed: cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min; samples blocking with 5% skimmed milk in PBS for 30 min [37 (link)]; primary antibodies (anti-NFκB, 1 : 200, Santa Cruz Biotechnology; anti-MyD800, Thermo Fisher Scientific; anti-DNMT1, 1 : 200, EpiGentek; and anti-p300, 1 : 200, OriGene) incubation for 2 h at room temperature; and finally, secondary antibody (Alexa Fluor 568 red fluorescence-conjugated goat anti-rabbit antibody, 1 : 200, Molecular Probes, Invitrogen, Eugene, OR, USA) incubation for 1 h at 37°C. Cells were stained for 1 h with Alexa Fluor 488 phalloidin green fluorescent conjugate (1 : 400, Molecular Probes) and for 1 h with TOPRO (1 : 200, Molecular Probes) to mark the cytoskeleton actin and nuclei, respectively [38 (link), 39 (link)]. The Zeiss LSM800 confocal system (Zeiss, Jena, Germany) has been used to acquire microphotographs.

After 5 days of 5-aza treatment, control and treated MUCs were fixed in 4% paraformaldehyde for 10 min at room temperature, followed by incubation in PBS containing 5% donkey serum (Jackson Immunoresearch), and 0.2% Triton X-100 (Sigma) for 20 min. Primary antibodies included epithelial markers anti-E-cadherin (1:200; Santa Cruz) and anti-pan-cytokeratin 26 (PCK26; 1:100; Sigma), and hair cell markers anti-Myosin VI (Myo6; 1:200; Sigma), anti-Myosin VIIa (Myo7a; 1:200; DSHB), anti-Math1 (Atoh1; 1:200; DSHB), anti-Pou4f3 (1:200; Sigma), anti-Espin (1:100; a gift from Dr. James Richard Bartles, Northwestern University), anti-DNMT1 (1:100; Epigentek) and anti-PCNA (1:100; Abcam). Secondary antibodies were Dylight-488-, Dylight-549,- and Dylight-649-conjugated antibodies, with a universal nuclear marker, 4′,6-diamidino-2-phenylindole (DAPI, all from Jackson Immunoresearch). MUC samples were incubated with primary antibodies overnight at 4°C, followed by secondary antibodies incubation for 2–3 h at room temperature. MUCs were observed using a Leica epifluorescence microscope and images were captured using a QImaging monochrome cool CCD camera.