Nis elements research br software

Cells were grown on poly-l-lysine coated glass coverslips and treated for 8h with the indicated drugs. Twelve minutes before the end of treatment, EdU was added to the media at a final concentration of 125 μM. Cells were fixed with 4% PFA for 10 min at RT, washed in PBS and stored overnight at 4 °C in fresh PBS and wrapped in foil. Cells were then permeabilized with 0.3% Triton X-100 in PBS for 3 min on ice. Following PBS washes, a Click-iT reaction was performed to tag the EdU alkyne using 10 μM biotin-azide, 2 mM CuSO₄ and 10 mM sodium ascorbate for 1 h at RT in a humidified chamber protected from light. Cells were then washed in PBS once before proceeding with the PLA assay between biotin-labeled nascent DNA and MCM6 following the manufacturer's instructions (Duolink, Millipore Sigma). Rabbit anti-biotin antibody (Bethyl Laboratories, #A150-109A; 1:3000) was used together with either mouse anti-MCM6 (Santa Cruz, sc-393618, 1:500) or mouse anti-biotin (Jackson ImmunoResearch, #200-002-211, 1:2000) to control for EdU uptake. Coverslips were mounted on slides and imaged with an Eclipse Ts2R-FL inverted Nikon fluorescence microscope equipped with a Nikon DSQi2 camera. Images were analyzed using the NIS-Elements Research BR software (Nikon) and quantifications were analyzed using Prism (GraphPad).

Coverslips were coated with poly-l-lysine (Millipore Sigma) for 15 min, and cells were seeded at least 24 h before any drug treatments. Following treatments, cells were washed once with cold PBS on ice and fixed with 4% paraformaldehyde for 10 min at RT. After three washes with PBS, cells were permeabilized with 0.3% Triton X-100 in PBS for 3 min on ice. Following permeabilization, cells were washed three times with cold PBS and blocked for 1 h at RT using 5% BSA in PBS. Subsequently, cells were incubated with primary antibodies diluted in 1% BSA for 2 h at RT. After three PBS washes, cells were incubated with secondary antibodies coupled to fluorophores diluted in 1% BSA for 45 min at RT. Following three additional washes with PBS, coverslips were mounted onto glass microscope slides using Vectashield mounting medium containing DAPI (Vector Lab). Coverslips were imaged with an Eclipse Ts2R-FL inverted fluorescence microscope (Nikon) equipped with a Nikon DSQi2 digital camera and analyzed using NIS-Elements, Research BR software (Nikon). Quantification data was processed using Prism (GraphPad).