Vectashield immunofluorescence medium

HEK293T cells were transiently transfected with constant amounts (1 μg) of plasmids encoding for 3xHA-D₂R^GFP2, 3xHA-D₂R^GFP2(Tyr192Ala^5.41x42) or 3xHA-D₂R^GFP2(Leu207Ala^5.56/Lys211Ala^5.60). Then, cells were fixed in 4% paraformaldehyde for 10 min, washed with PBS containing 20 mM glycine, and mounted in a Vectashield immunofluorescence medium (Vector Laboratories, United Kingdom). Microscope observations were performed with a 40× oil immersion objective in a Leica TCS-SL confocal microscope (Leica, United States).

Internalization of D₂R were evaluated by fluorescence confocal microscopy using transiently single or cotransfected HEK293T cells with constant (1 μg) amounts of plasmids encoding for Sigma1R and D_2LR^YFP. Cells were incubated with cocaine (100 nM) at different interval times or with a range of cocaine concentrations (1 nM–10 μM) during 30 min. Then, cells were fixed in 4% paraformaldehyde for 10 min, washed with PBS containing 20 mM glycine, and mounted in a Vectashield immunofluorescence medium (Vector Laboratories, UK). Microscope observations were performed with a × 63 oil immersion objective in a Leica TCS-SL confocal microscope (Leica, USA). The amounts of internalized D₂R^YFP are shown as a single z-scan image. The rate of internalization was measured as the ratios (IntROI/membROI). The memROI is obtained by measuring the D₂R^YFP fluorescence area detected at the cell surface membrane depicted by phase contrast). The IntROI is instead obtained by measuring the entire D₂R^YFP fluorescence area detected in the cytoplasm of the entire cell. The basal value is obtained from cells not exposed to cocaine.