Ab 881796

PLA was used for the in situ detection of the interaction between D1 and D2 receptors and also between σ1Rs and IP₃R1s. The assay was performed in a humidified chamber at 37 °C according to the instructions of the manufacturer (Olink Bioscience, Sweden). For this method, following antibodies were used: rabbit polyclonal antibody to OPRS1 (σ1R; AB_881796, Abcam, UK), mouse monoclonal antibody to IP₃R1 (AB_212025, Calbiochem, Merck Biosciences, Germany), mouse monoclonal antibody to dopamine receptor D1 (SG2-D1α, ab78021, Abcam, UK), and rabbit polyclonal antibody to dopamine receptor D2 (ab21218, Abcam, UK).

The appropriate monoclonal (3 µg) or polyclonal antibody (6 µg) was incubated with 60 µl of washed magnetic beads (Dynabeads M-280 coated with sheep anti-mouse IgG or M-280 coated with sheep anti-rabbit IgG (Life Technologies, Dynal AS, Norway)) overnight at 4 °C on a rotator (VWR International, LLC, PA, USA). The beads with attached antibodies were washed twice (200 µl) with phosphate-buffered saline (PBS supplemented with 1% bovine serum albumin). Proteins were immunoprecipitated from 1 mg of detergent-extracted total protein via their incubation with antibody-bound beads for 4 h at 4 °C. Bead complexes were washed with PTA (4× with 200 µl; 145 mmol/L NaCl, 10 mmol/L NaH₂PO₄, 10 mmol/L sodium azide, and 0.5% Tween 20; pH 7.0). Immunoprecipitated proteins were then extracted with 60 µl of 2× Laemmli loading buffer according to the manufacturer’s instructions (Bio-Rad) and boiled for 5 min. The following antibodies were used for immunoprecipitation: rabbit polyclonal antibody to OPRS1 (σ1R; AB_881796, Abcam, UK) and mouse monoclonal antibody to IP₃R1 (AB_212025, Calbiochem, Merck Biosciences, Germany).

Cells grown on glass coverslips were fixed in ice-cold methanol. Nonspecific binding was blocked by incubation with PBS containing 3% bovine serum albumin (BSA) for 60 min at 37 °C. The cells were then incubated with primary antibody diluted 1:500 in PBS with 1% BSA (PBS–BSA) for 1 h at 37 °C. A rabbit polyclonal antibody (AB_212026, Calbiochem, Merck Biosciences, Darmstadt, Germany) directed against 1829–1848 amino acid residues from human IP₃R1 was used. Another group of cells was incubated with rabbit polyclonal antibody anti-OPRS1 (AB_881796, Abcam, USA) directed against a synthetic peptide derived from the C-terminal region of rat σ1 peptide. Afterwards, the cells were washed three times with PBS/BSA for 10 min, incubated with CF488A goat anti-rabbit IgG (AB_10559670, Biotium) diluted 1:1000 in PBS/BSA for 1 h at 37 °C, and washed as described previously. Finally, the cells were mounted onto slides in mounting medium with Citifluor (Agar Scientific Ltd., Essex, UK) and analyzed by laser scanning confocal microscopy (LSM 510 MetaMicroscope, Zeiss). Images were taken with a Plan Neofluar 40×/1.3 oil objective. Images were scanned at scan speed 7 (260 Hz line frequency), 1024 × 1024 pixels, 12 bit data depth in the average mode (4× line) at optical zoom 3. The Z-stack interval was 0.8 µm. Images of all samples were acquired with the same microscope setup.