Duolink probemaker

Brain slices were prepared as previously described^{21 (link)} and proximity ligation assay (PLA) was performed using the Duolink in situ kit (Olink Bioscience, Uppsala, Sweden) according to the manufacturer's instructions with the following modifications: PLA probes incubation was for 2 h; ligation was performed for 45 min; amplification step was extended for 2 h with a concentration of polymerase of 1/60 all at 37°C. Striatal primary cultured cells were plated into 8-μ well plates (LabTek, Dutscher, Brumath, France). Blocking (1 h at room temperature) and primary antibody (overnight at 4 °C) incubations were performed in a 3% bovine serum albumin (Sigma Aldrich) and 0.2% Triton X-100 solution. Rabbit anti-GluN1 (ab17345, Abcam, Cambridge, MA, USA) and rat anti-D1R (D2944, Sigma Aldrich) were diluted (1:500) in the blocking solution. The anti-rabbit (+) PLA probe (1:5) along with an anti-rat (−) probe (1:100) were diluted in the blocking solution. Anti-rat PLA probes were generated according to the manufacturer's instructions using the Duolink Probemaker (Olink Bioscience). Goat-anti-rat IgGs (Santa Cruz, Heidelberg, Germany) were used. Cells were mounted in FluorSave (Calbiochem). Image acquisition and quantification are detailed in the Supplementary Information section.

The proximity ligation assay was performed using the Duolink In Situ Kit (Olink Bioscience, Uppsala, Sweden) according to the manufacturer's instructions with the following modifications: PLA probe incubation was of 2 h; amplification step was extended to 2 h. Blocking (1 h at room temperature) and primary antibody (overnight at 4 °C) incubations were performed in a 4% bovine serum albumin (Sigma Aldrich) and 0.2% Triton X-100 solution. Rabbit anti-GluN1 (ab17345, Abcam, Cambridge, MA, USA) and rat anti-EGFR (ab231, Abcam) were diluted (1 : 2000 and 1 : 100, respectively) in the blocking solution. The anti-rabbit (+) PLA probe (1 : 5) along with an anti-rat (−) probe (1 : 100) were diluted in the blocking solution. A Goat anti-rat (Jackson ImmunoResearch Inc., Suffok, UK) were used to make a probe anti-rat according to the manufacturer's instructions using the Duolink Probemaker (Olink Bioscience). Slides were mounted in a mounting medium containing DAPI (4′,6-diamidino-2-phenylindole) and 0.1% deparaphenylene-diamine diluted in phosphate-buffered saline and glycerin. The negative control represents the PLA without the primary antibodies.
Ten stack of picture (0.40 μm per section) were taken from 10 different areas of every well with a confocal microscope (Leica SP5, Leica, Nanterre, France). Punctuas were counted manually using a z projection of the stack.