Eclipse ts2r fl inverted

Cells were seeded on coverslips at least 24 h before any drug treatment. To stop the treatment, cells were washed with cold PBS on ice, then fixed with 4% paraformaldehyde for 10 min at 4 °C. 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 PBS, and blocked for 45 min at RT using 5% BSA in PBS. Subsequently, cells were incubated with primary antibodies diluted in 1% BSA for 1 h at RT. After three PBS washes, cells were incubated with secondary antibodies coupled to fluorochromes diluted in 1% BSA for 45 min at RT. After three additional PBS washes, coverslips were mounted onto microscope slides using Vectashield mounting medium containing DAPI (Vector Lab). Coverslips were analyzed with an Eclipse Ts2R-FL inverted Nikon fluorescence microscope equipped with the Nikon DSQi2 digital camera. Fluorescence images were analyzed using Fiji, and quantification data was processed by Prism (GraphPad).

Cells were grown on coverslips and incubated with 10 µM BrdU for 48 h before HU treatment at 2 mM for 4 h. Cells were permeabilized with 0.3% Triton X-100 in PBS for 3 min at 4 °C, washed three times in PBS, and fixed with 4% paraformaldehyde for 10 min at 4 °C. After three PBS washes, cells were blocked with 5% BSA, then incubated with 1:300 mouse anti-BrdU (BU-1) antibody in 1% BSA for 2 h. After three PBS washes, cells were incubated with 1:1,000 Alexa Fluor 488 goat anti-mouse IgG for 45 min in 1% BSA. After three PBS washes, coverslips were mounted using Vectashield mounting medium containing DAPI (Vector Lab) and analyzed with the Eclipse Ts2R-FL inverted Nikon fluorescence microscope equipped with the Nikon DSQi2 digital camera. Corrected total cell fluorescence (CTCF) intensity was quantified and calculated using Fiji and analyzed with Prism (GraphPad).