Biocoat poly l lysine coated glass coverslips

Sixty thousand SUM159 cells were plated on BioCoat Poly-l-Lysine-coated glass coverslips (Corning, Corning, NY) and allowed to adhere for 18 h. Cells were treated with 50 µM fisetin for 30 min at 37 °C. Media was removed, and cells were fixed in 4% paraformaldehyde for 15 min at room temperature. Cells were blocked and permeabilized 1 h at room temperature in 1xPBS containing 5% goat serum (Sigma-Aldrich) and 0.3% triton X-100 (Fisher Scientific) before primary antibody incubation of fibrillarin (1:500, Abcam ab4566, Cambridge, UK) in 1xPBS containing 1% bovine serum albumin (BSA; Fisher Scientific) and 0.3% triton X-100 for 1 h at room temperature. Goat-anti-mouse AlexaFluor 594 (Thermo Fisher; A21125) secondary antibody incubation was as for primary. Coverslips were washed three times in 1xPBS before being mounted on microscope slides using ProLong Diamond Antifade Mountant (Thermo Fisher). Images were captured using a Nikon A1plus. Digital deconvolution and documentation of images were achieved using NIS Elements software (Nikon).