Cells were prepared as per immunofluorescence as above. Two primary antibodies selected from two different host species (rabbit anti-plek2, 1:100 (Proteintech); mouse anti-Rac1, 1:250 (BD Bioscience, 610650)) are used together with the generic Duolink species-specific secondary antibodies containing unique DNA strands that template the hybridization of added oligonucleotides. When in close proximity (<40 nm), the oligonucleotides are ligated by a ligase to form a circular template. This template, still anchored to the antibody, is subsequently amplified and detected using complementary labeled oligonucleotide probes62 (link)–64 (link). The resulting distinct spots were derived from single-molecule protein interaction events.
Quantifications of PLA mean fluorescence intensity per cell was performed by using ImageJ software. PLA was performed using Duolink® In Situ Red Starter Kit Mouse/Rabbit (Sigma, DUO92101-1KT), according to the manufacturer’s instructions. Final resultant images were visualized using TCS SP5 confocal microscope (Leica Microsystems Heidelberg, Manheim, Germany) equipped with an Apo CS Lambda Blue 63 × 1.40 NA oil objective. Images (1024 × 1024) were acquired at 700 Hz in frame sequential mode, 2.7 zoom.
Quantifications of PLA mean fluorescence intensity per cell was performed by using ImageJ software. PLA was performed using Duolink® In Situ Red Starter Kit Mouse/Rabbit (Sigma, DUO92101-1KT), according to the manufacturer’s instructions. Final resultant images were visualized using TCS SP5 confocal microscope (Leica Microsystems Heidelberg, Manheim, Germany) equipped with an Apo CS Lambda Blue 63 × 1.40 NA oil objective. Images (1024 × 1024) were acquired at 700 Hz in frame sequential mode, 2.7 zoom.