Rabbit anti pcna antibody

Cells were grown on coverslips, washed with PBS, fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.2% Triton X-100 for 5 min. Cells were then blocked in 3% BSA, 0.1% Tween-20 in 4XSSC for 1 h at room temperature. Cells were then incubated with primary antibody overnight at 4 °C [1:500 goat anti-RNA polymerase II antibody (PLA0292, Sigma) with 1:500 rabbit anti-PCNA antibody (PLA0079, Sigma)]. The next day after washing with 1 ×  PBS twice, cells were incubated with pre-mixed PLA probe anti-goat minus and PLA probe anti-rabbit plus (Sigma) for 1 h at 37 °C. The subsequent steps in proximal ligation assay were carried out with Duolink In Situ Kit (Sigma) in accordance to manufacturer’s instructions. In brief, cells were washed with Buffer A for 5 min three times, ligation reaction for 30 min at 37 °C, washed with Bufer A for 2 min two times, and Amplification for 100 min at 37 °C. After washing with Buffer B for 10 min three times and 0.01% Buffer B for 1 min, slides were then stained with DAPI and imaged on LeicaDM18 microscope at ×100. Negative controls were treated identically but anti-RNA polymerase II antibody was omitted.