Dm 14000b confocal microscopy

Colon cancer cells were seeded onto coverslips in a six-well plate and fixed with 4% paraformaldehyde (w/v) for 30min. Coverslips with colon cancer cells were then washed with PBS for 15 min and permeabilized with 0.2% (w/v) Triton X-100 in PBS for 5min. PBS containing 1% bovine serum albumin (BSA) was used to block for 30 min. Coverslips were subsequently incubated with the primary antibodies against XRCC5 or p300 diluted in PBS containing 10% BSA overnight. Coverslips were washed with PBS for 3 times and then incubated with secondary fluorescein isothiocyanate conjugated antibody or tetra methyl rhodamine isothiocyanate conjugated antibody for 1 hour. After washed with PBS for 3 times, coverslips with colon cancer cells were stained with DAPI (Beyotime). Leica DM 14000B confocal microscopy was employed to detect fluorescence and localize XRCC5 and p300 expressions in cells.

The Cells were seeded onto coverslips in a 6-well plate and fixed with 4% paraformaldehyde (w/v) for 30 mins, and then were washed for 10 mins with PBS and permeabilized with 0.2% (w/v) Triton X-100 in PBS for 5 mins. The blocking step was performed for 30 min in PBS containing 1% bovine serum albumin (BSA). Cells were then incubated overnight with the primary Ku80 or CBP antibodies diluted in PBS containing 10% BSA. After being washed with PBS, cells were incubated for 1 h with secondary fluorescein isothiocyanate or tetra methyl rhodamine isothiocyanate-conjugated antibodies. After several additional washing steps, the cells were stained with DAPI (Beyotime, China). The localization of Ku80 and CBP protein was assessed using a Leica DM 14000B confocal microscopy.