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, A2AR 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.
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, A2AR 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.
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