Rabbit anti pcna d3h8p

A proximity ligation assay (PLA) was performed with Duolink in situ detection reagents orange (Sigma Aldrich) according to the manufacturer’s recommendations, with minor modifications. Neutrophils were settled onto coverslips coated with poly-l-lysine (Corning) for 30 min at 37°C, and infections were done for 16 h. Cells were fixed with 4% PFA for 15 min at RT, washed with PBS, and blocked with Duolink in situ blocking solution for 30 min at 37°C. Cells were then stained with mouse anti-caspase-3 [3CSP03(4.1.18), 1:100; Invitrogen] and rabbit anti-PCNA (D3H8P, 1:200; Cell Signaling) primary antibodies for 4 h at RT. Cells were washed and stained with the secondary mouse PLUS and rabbit MINUS antibodies for 1 h at 37°C. Cells were washed in Duolink in situ wash buffer, the ligation reaction was performed at 37°C for 30 min, and it was followed by the amplification reaction at 37°C for 100 min. Cells were washed in Duolink in situ wash buffer and stained with Hoechst 33342 (ImmunoChemistry Technologies) for 5 min at 37°C. Coverslips were mounted onto glass slides using the Duolink in situ mounting medium with DAPI. Images were acquired using a Leica SP8 confocal microscope with a 63× oil objective. Images were analyzed using FIJI (81 (link)).