Plan apochromat 63 1.4 numerical aperture oil objective

HeLa cells were nucleofected with AG49CMVGag-RTEm26CTE plasmid expressing NHL-a101 or NHL-a102 (HeLa-NHL-a101 and HeLa-NHL-a102, respectively), cultured for 24 h in complete medium, and then stained for confocal microscopy analysis as follows: fixed with 3% paraformaldehyde/2% sucrose in PBS for 30 min, permeabilized with 0.5% Triton, and saturated with 1% BSA in PBS. Then, the cells were incubated for 1 h with the anti-p17 mAb MBS-3 followed by Alexa Fluor 488–conjugated antimouse IgG (Molecular Probes) at room temperature. When indicated, not fixed cells were saturated with 1% BSA in PBS, incubated for 1 h with the anti-p17 mAb MBS-3 followed by Alexa Fluor 488–conjugated antimouse IgG on ice and then fixed as aforementioned. Nuclei were counterstained with DAPI (Sigma). Cells were analyzed using a Zeiss LSM510 Meta confocal microscope equipped with a Plan-Apochromat 63×/1.4 numerical aperture oil objective. The excitation sources were an argon ion and 405-nm diode lasers for Alexa Fluor 488 and DAPI, respectively. Alexa Fluor 488 was acquired using a LP 505 filter, whereas DAPI was acquired using a LP 420 combined with an NFT 490 beam splitter. Single images and orthogonal projection from z-stack sections were obtained through Zen Black 2 software (Zeiss).

In situ proximity ligation assay (PLA) was carried out in HEK-D3R-nAChR transfected cells by using the Duolink in situ detection kit (Sigma-Aldrich) according to the manufacturer’s instructions, as previously reported [3 (link), 4 (link)]. Briefly, cells were fixed in PBS containing 4% paraformaldehyde (Sigma-Aldrich) for 10 min at room temperature, washed in PBS and blocked with blocking solution (Duolink, Sigma-Aldrich) for 30 min at 37 °C. Cells were next incubated overnight at 4 °C with the goat polyclonal anti-D3R (1:50; Santa Cruz Biotechnologies) and the rabbit monoclonal anti-alpha4 nAChR subunit (1:100; Sigma-Aldrich) antibodies. After washing, cells were incubated with the anti-rat MINUS and the anti-goat PLUS probes (Duolink, Sigma-Aldrich) for 30 min at 37 °C, followed by ligation and amplification reaction using Duolink far red detection reagent. Cells were then mounted using mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; Duolink, Sigma-Aldrich) and analyzed using a Zeiss LSM 880 confocal microscope equipped with Plan-Apochromat 63 × /1.4 numerical aperture oil objective and examined using the ZEN 2.3 software (Carl Zeiss AG, Oberkochen, Germany).