Retiga r6 monochrome camera

Cells were seeded on cover slide in 12-well plate and fixed with 4% paraformaldehyde in room temperature for 15 min. Afterward, cells were permeabilized with pre-cooled 0.5% TritonX-100 (dissolved in PBS) on ice for 5 min. After blocking with 1% bovine serum albumin, cells were incubated with primary antibodies at room temperature for 1 h. Primary antibodies used are anti-FLAG (Sigma #F7425), anti-Myc tag (Santa Cruz #SC-40), anti-BRD4 (CST #13440) and anti-USP1 (Proteintech #14346-1-AP). Slides were then incubated with secondary antibodies for 1 h at room temperature (Invitrogen #715-545-150, #711-585-152, and #711-545-152). Nuclei were counterstained with 1.5 μM 4′,6-diamidino-2-phenylindole for 5 min. Finally, cells were mounted onto a slide with mounting medium (Abcam #AB104135). Photos were taken with QImaging Retiga R6 Monochrome camera connected to Zeiss-A1 Axiovert A1 fluorescence microscopy equipped with 63 × oil object lens.

Immunofluorescence was performed as we described previously (42 , 89 ). Cells were seeded on sterile microscope cover glass precoated with 0.2% gelatin (Sigma #G1890) dissolved in ultrapure water. Cells were rinsed with PBS, fixed with 4% formaldehyde for 15 min at room temperature, washed with PBS for three times and next permeabilized with precooled 0.5% Triton X-100 in PBS. Cells were then blocked with 1% bovine serum albumin for 30 min and incubated with primary antibodies for 1 h at room temperature. Anti-HA tag antibody (Cell Signaling Technology #3724) was used in immunofluorescence. Afterward, cells were washed with PBS containing 0.2% Tween-20 for three times and incubated with Alexa Fluor–conjugated secondary antibody (Jackson ImmunoResearch #711-585-152) for 1 h in dark at room temperature. The 4′,6-diamidino-2-phenylindole was used to counter-stain cell nuclei at 1.5 μM final concentration. Finally, cover glass was mounted to a slide with antifading fluoromount-G mounting medium (Invitrogen #00-4958-02). Images were taken with QImaging Retiga R6 monochrome camera connected to Zeiss Axiovert A1 fluorescence microscope.