Duolink proximity ligation assay kit

Interaction was detected by a Duolink Proximity Ligation Assay kit (Olink Bioscience, Uppsala, Sweden: PLA Probe anti-Mouse MINUS; PLA Probe anti-Rabbit PLUS; Detection Kit 563). The PLA Probe anti-Mouse MINUS binds to the HA antibody (Cell Signaling; #2367), whereas the PLA Probe anti-Rabbit PLUS binds to the FLAG antibody (Cell Signaling; #2368). After preincubation with a blocking agent for 1 h, the fixed HEK293T cells were incubated overnight with the primary antibodies to anti-HA (Cell Signaling, 1:100) and anti-FLAG (Cell Signaling, 1:100). Duolink PLA probes detecting mouse or rabbit antibodies were diluted in the blocking agent to a concentration of 1:5 and applied to the slides, followed by incubation for 1 h in a pre-heated humidity chamber at 37 °C. Unbound PLA probes were removed by washing. The slides were then incubated in a ligation solution consisting of Duolink Ligation stock (1:5) and Duolink Ligase (1:40) for 30 min at 37 °C. Detection of the amplified probe was performed with the Duolink Detection kit. Duolink Detection stock was diluted at 1:5 and applied for 100 min at 37 °C. Final washing steps were carried out in saline sodium citrate buffer. Duolink PLA signals were observed with an Olympus Fluoview FV1000 confocal microscope (Olympus) at room temperature.