Cy5 conjugated anti rabbit secondary antibody

HP1β was detected with a mouse monoclonal antibody (clone 1 MOD 1A9, Euromedex, 1:250 diluted in 2% bovine serum albumin (BSA) in PBS), using a rhodamine (TRITC)-conjugated anti-mouse secondary antibody (#715–025-151, Jackson ImmunoResearch, USA). The centromeres were labeled with a human CREST antibody which mostly recognizes CENP-A (Immunovision, Cellon Sarl, 1:250 in 2% BSA/PBS), using FITC-conjugated anti-human secondary antibody (#709–095-149, Jackson ImmunoResearch, USA). H3K9me3 and H4K20me3 were detected with rabbit polyclonal antibodies (39161 from Active Motif and ab9053 from Abcam, diluted 1:500 in 2% BAS/PBS) using a Cy5-conjugated anti-rabbit secondary antibody (#711–175-152, Jackson ImmunoResearch, USA). All the Jackson ImmunoResearch secondary antibodies raised in donkey were used at a dilution of 1:200 in 2% BSA/PBS.

Cells were grown on coverslips preplaced in the 6-well culture plates. After 24 h cultivation, cells were fixed with 4% paraformaldehyde, and then permeabilizated with 0.2% Triton X-100 in PBS for 30 min. Then, the cells were blocked with 10% goat serum for 1 h at room temperature. Immunofluorescence staining was determined by Dcp1a and EDC4 primary antibodies at 4 °C overnight. After washing three times with PBS, cells were incubated with the Cy5-conjugated anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at room temperature. The nuclei of cells were counterstained with Hoechst 33,342 (Invitrogen, Carlsbad, CA, USA) for 1 min and washed with PBS. The stained cells were mounted using Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL, USA), and subsequently observed using a Carl Zeiss LSM 710 confocal microscope system.