Adherent HeLa cells on the glass slides were transfected with the indicated plasmids. After fixation, cells were pretreated and blocked with 5% BSA. Primary antibodies against GFP and Syk were used for the slides. The secondary antibodies, which were conjugated with oligonucleotides (PLA probe MINUS [Sigma no. DUO92004] and PLA probe PLUS [Sigma no. DUO92002]), were added to the glass slides for 1 h at 37°C. After incubation, a system consisting of two oligonucleotides and a ligase were added to the system for 30 min at 37°C. If the oligonucleotide probes were in close proximity, the ligation reaction between them formed a closed DNA circle. Then, the slides were incubated for amplification by the PLA probe (Sigma no. DUO92008) and the polymerase for 100 min at 37°C. The amplification reaction results in a signal which was seen as a unique fluorescent spot under confocal microscopy. The PLA dots were counted via Fiji software and analyzed as previously described (40 (link)).
Free full text: Click here
