Lsm500 confocal microscope

Cells were fixed with 2% paraformaldehyde in cell growth medium for 15 min and permeabilized with permeabilization buffer (0.05% Triton-X in PBS) for 5 min three times at room temperature. Cells were blocked with blocking buffer (Universal blocking buffer, BiogeneX) for 45 min and then incubated with multiple combinations of primary antibodies including anti-α-actinin (Mouse monoclonal, Sigma, 1:400 dilution), anti-GFP (Chicken IgY fraction, Invitrogen, 1:400 dilution), anti-tagRFP (Rabbit polyclonal, Evrogen, 1:400 dilution, for detecting tagBFP), and anti-mCherry (Rat monoclonal, Thermo Fisher Scientific, 1:1000 dilution) for 1.5 hours at room temperature. Following washing three times for 5 min with permeabilization buffer, cells were incubated with appropriate Alexa fluorogenic secondary antibodies (Invitrogen) at 1:400 dilution and DRAQ5 for nuclear staining (Abcam) at 1:500 dilution at room temperature for 1 hour. After another set of washing (5 min x3 with permeabilization buffer), cell images were captured with Zeiss LSM 500 confocal microscope or Olympus IX81 epifluorescent microscope. Alternatively, Hoechst (Invitrogen) staining was used to visualize cellular nuclei. For Hoechst nuclear staining, Hoechst solution was added in washing buffer at final concentration of 2 µM for 15 min after first washing following secondary antibody incubation.