Chromatin-bound Replisome Visualization

Cells were immediately permeablized and fixed after EdU labeling. In brief, cells were extracted with CSK buffer (10 mM Hepes, 300 mM Sucrose, 100 mM NaCl, 2 mM MgCl₂, and 0.5% Triton X-100, pH = 7.4) for 10 minutes to remove replisome proteins that are unbound to chromatin, followed by fixation with paraformaldehyde (PFA, 4% in PBS) for 30 minutes24 (link)25 (link). Cells were then washed twice with blocking buffer (2% glycine, 2% BSA, 0.2% geltin, and 50 mM NH₄Cl in PBS) and blocked for 1 hour at room temperature or overnight at 4 °C for further immunofluorescence staining.
Nascent DNA staining was performed by tagging Alexa Fluor 647 picolyl azide onto EdU via the ‘click’ reaction26 (link) (Click-iT chemistry, thermoFisher C10640). RPA, PCNA, and MCM were stained with validated monoclonal antibodies27 (link)28 (link)29 (link). In detail, RPA was immunostained with a rabbit monoclonal against RPA70 (Abcam, ab79398) followed by secondary immunostaining with goat-anti-rabbit conjugated with Alexa Fluor 568 (ThermoFisher, A11036); PCNA and MCM were immunostained with Mouse monoclonal against PCNA (Abcam, Ab29) and MCM5 (Abcam, ab6154), respectively, followed by secondary immunostaining with goat-anti-mouse conjugated with alexa Fluor 488 (ThermoFisher, A11029).
Coverslips were mounted onto a microscope microfluidics chamber for SR imaging.