Flow cytometry analysis of PCNA in HeLa cells. Cells were harvested, adjusted to a concentration of 1 x 10^6 cells/mL, and fixed and permeabilized with 90% methanol. In the left panel, cells were blocked with 1% BSA in PBS (Product # 37525), and stained with a biotinylated PCNA monoclonal antibody (Product # MA5-11355) at a 1:500 dilution in blocking buffer (blue histogram) or incubated in blocking buffer alone as a negative control (red histogram), followed by Streptavidin-DyLight 550 (Product # 84542) at a dilution of 1:1000 for 30 minutes at room temperature. In the right panel, cells were blocked with Solution 1 from a biotin-streptavidin blocking kit (to block endogenous biotin), and stained with a biotinylated PCNA monoclonal antibody (Product # MA5-11355) at a 1:500 dilution in blocking buffer containing Solution 2 from a biotin-streptavidin blocking kit (blue histogram) or incubated with blocking buffer plus Solution 2 (red histogram), followed by Streptavidin-DyLight 550 (Product # 84542) at a dilution of 1:1000 for 30 minutes at room temperature. A representative 1,000 cells were acquired for each sample. Unstained cells are shown by the black histograms in each panel. NOTE: Blocking endogenous biotin is required to eliminate background signal.