Mabn53

Sections were treated with 10 mM citrate (pH 6) of 65 °C at room temperature (RT) for 30 min. After three washes with PBS (5 min each), 10 mM glycine (in PBS) was applied for 20 min, followed by two washes with PBS (5 min). Sections were then permeabilized with 0.3% Triton X-100 for 30 min, washed twice with PBS (5 min) and pre-incubated in blocking buffer (SuperBlock®, ThermoFisher Scientific Prod# 37,515) for 30 min. Subsequently, they were incubated under gentle agitation at 4 °C overnight with each of the following primary antibodies: mouse monoclonal anti-KOR (1:25, 1:50) (ab201552, abcam), anti-Tyrosine hydroxylase Alexa Fluor 488 (1:100) (MAB318-af488, Millipore), rabbit anti-D4R (orb39453, Biorbyt), rabbit anti-D1R (orb107494, Biorbyt), mouse anti-D2R (1:600) (MABN53, Millipore) and rabbit monoclonal anti-SSTR2 (1:100) (ab134252, abcam). After two washes (5 min) with 1:2 dilution of blocking buffer in PBS, slices were incubated for 2 h at 37 °C with the secondary antibodies Alexa Fluor 488-conjugated goat anti-mouse (A11001) or anti-rabbit (A11034, A11008) (1:200, Invitrogen). Following two washes with the diluted blocking buffer in the dark, sections were mounted on SuperFrost glass slides with the addition of mounting medium (Duolink® In Situ Mounting Medium with DAPI, DUO82040, Sigma-Aldrich) and stored at − 20 °C.

In situ proximity ligation assay (PLA) in cultured cells was performed using the Duolink in situ PLA detection kit (Sigma-Aldrich, St. Louis, MO, USA), following the protocol described previously [11 , 37 , 38 (link)] using mouse monoclonal anti-D₂R (2 μg/ml, MABN53; Millipore, Billerica, MA, USA) and rabbit polyclonal anti-mGluR₅ (2 μg/ml, AB5675; Millipore) primary antibodies. PLA control experiments employed only one primary antibody. The PLA signal was visualized and quantified by using a TCS-SL confocal microscope (Leica Lasertechnik GmbH, Heidelberg, Germany) and the Duolink ImageTool software. High magnifications of the microphotograph were taken and visualized using multiple z-scan projections.
The background signal was estimated from both PLA control experiments and from PLA experiments performed on non-transfected HEK293T cells (HEK293T cell line expresses endogenously small amount of D₂R, A_2AR and mGluR₅). In general, the positive PLA values obtained in these experiments were residuals. The assay cut-off value was set to two standard deviations over the background signal. Therefore, samples with values below this cut-off were negative for the interaction of interest, while samples with values higher than the threshold were positive.