Duolink 2

PLA is a sensitive and specific in situ protein-protein interaction assay where signals generated by primary and secondary antibodies are amplified by rolling circle replication of circular DNA generated by antibody-liked oligonucleotides, followed by fluorescent labeling to produce punctate fluorescent dots (50 (link)). PLA was performed using the Duolink II (Olink Bioscience)/ Millipore-Sigma reagents and protocol.

In situ PLA was performed using Duolink II (Olink Bioscience, Uppsala, Sweden) as described previously (Woskowicz et al., 2013 (link)). HEK293 stable cell lines or A431 cells were cultured on gelatin-coated cover glasses and fixed with 3% paraformaldehyde/PBS for 10 min at room temperature, followed by 30 min of incubation with blocking solution (Duolink II) at 37°C. After blocking, cells were immunostained with 20 μg/ml anti-DDR1 ectodomain goat antibody and 10 μg/ml anti-ADAM10 ectodomain mouse antibody for 2 h at room temperature, followed by 1 h of incubation at 37°C with a secondary antibody to goat immunoglobulin G (IgG) conjugated with PLA probe PLUS and a secondary antibody to mouse IgG conjugated with PLA probe MINUS. For detection of PLA signals, Duolink II detection reagents orange was used. The PLA signals were observed by wide-field TE2000-E microscope equipped with a Hamamatsu Orca ER charge-coupled camera (Hamamatsu, Hamamatsu, Japan) with a Nikon Plan Fluor 10× dry lens with numerical aperture (NA) 0.3 (Nikon, Melville, NY) and Volocity acquisition software (PerkinElmer-Cetus, Waltham, MA). The PLA signal is visible as a distinct fluorescent spot when both probes are in close proximity of <40 nm. The nucleus was counterstained with 4′,6-diamidino-2-phenylindole (DAPI). For washing steps, Duolink II Wash Buffer A and B (Olink Bioscience) were used.

To analyze the NRP1/PD1 interaction both in mice and in human, we used the Duolink II technology (Olink Bioscience), which is an in-situ proximity ligation assay technology. Slides were incubated with primary antibodies (anti-PD1 and anti-NRP1) and with secondary antibodies conjugated with oligonucleotides (PLA probe Minus anti-rabbit and PLA probe PLUS anti-mouse or goat). Ligation and amplification reactions were performed according to manufacturer’s instructions. Two types of negative controls were used: secondary antibodies alone and rabbit anti-IRAP antibody clone D7C5 (Cell signaling Technology).

Cells were fixed on slides using poly-L-lysine, and Duolink (Duolink II; Olink Biosciences) assay was performed according to the manufacturer’s instructions. In brief, cells were fixed with formaldehyde 2% for 15 min, permeabilized with Triton X-100 1% for 10 min, and incubated with RUNX1 (1/1,000) and RAG1 (1/25) antibodies for 1 h at room temperature. Images were collected on a confocal microscope (LSM 700; Carl Zeiss) with Zen 2011 software using 63× objectives at room temperature. Images were processed using ImageJ (National Institutes of Health).