Sp2 confocal microscope

The NIPA1 (NM_144599.4) tagged with FLAG in C-ter and cloned in pcDNA3.1 vector was acquired from GenEZ™ ORF cDNA Clones (GenScript, Piscataway, NJ, USA). The NIPA1 p.P91R and p.G106R missense mutations were introduced using a Site-Directed Mutagenesis kit (Agilent, Santa Clara, CA, USA) using specific primers for each mutation (available upon request). HeLa cells were grown in complete DMEM and transiently transfected with 2 µg of NIPA1 wild-type (WT), p.P91R, or p.G106R with FuGENE HD Transfection Reagent (Promega, Madison, WI, USA). After 24 h, the cells were fixed, permeabilised and blocked as previously described [13 (link)]. The samples were incubated with the primary antibodies anti-FLAG (Sigma-Aldrich, Saint Louis, MO, USA) and anti-Na⁺/K⁺-ATPase, anti-mannose-6-phosphate receptor (M6PR), anti-glucose-regulated protein 94 KDa (GRP94), or anti-early endosome antigen 1 (EEA1; Abcam, Cambridge, UK). The following day, they were exposed to the appropriate secondary antibodies conjugated with fluorophores (Invitrogen, Carlsbad, CA, USA) and examined using the SP2-Leica confocal microscope (Leica, Wetzlar, Germany).

Rat kidneys were snap-frozen and embedded in Tissue-Tek^® O.C.T. Compound (Sakura). Cryosections were fixed with 3.5% PFA, permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) at RT for 15 min and blocked with CAS-block (Invitrogen). Sections were incubated with rabbit anti-CD2AP (Santa Cruz Biotechnology), quinea pig anti-nephrin IgGs (Sigma Aldrich) and goat anti-SHIP2 I-20 IgGs (Santa Cruz Biotechnology) overnight at +4 °C in ChemMate (Dako Cytomation, Glostrup, Denmark), followed by Alexa Fluor 555 donkey anti-rabbit, Alexa Fluor 488 donkey anti-quinea pig and Alexa Fluor 555 donkey anti-goat IgGs (Invitrogen) together with Hoechst (Fluka, Sigma Aldrich) for 1 h in ChemMate (Dako Cytomation). Samples were mounted in Mowiol and examined with Leica SP2 confocal microscope (Leica Microsystems CMS GmbH, Mannheim, Germany).

For immunocytochemistry, HEK-293T cells stably expressing CB₁R were grown on glass coverslips and were transiently transfected with the corresponding cDNA. After 48 h of transfection, cells were fixed in 4% paraformaldehyde for 15 min and washed with phosphate-buffered saline (PBS) containing 20 mM glycine to quench the aldehyde groups. After permeabilization with PBS-glycine containing 0.05% Triton X-100 for 5 min, cells were incubated 1 h at room temperature with PBS containing 1% bovine serum albumin and were labeled overnight with the corresponding primary antibody: guinea pig anti-CB₁R (Frontier Science, Ishikari, Japan) or rabbit anti-CB₁R antibody (Thermo Scientific, Fremont, California), rabbit anti-5-HT_2AR antibody (Neuromics, Edina, Minnesota), mouse anti-transferrin antibody (Abcam, Cambridge, UK) or guinea pig anti-D₁R antibody (Frontier Science, Ishikari, Japan); washed, and stained 2 h with the secondary antibody: chicken anti-rabbit (1:200, Alexa Fluor 594, Invitrogen), goat anti-guinea pig (1:200, Alexa Fluor 488, Invitrogen), or goat anti-mouse (1:200, Alexa Fluor 488, Invitrogen). Samples were rinsed several times and mounted with Mowiol medium (30% Mowiol, Calbiochem, Darmstadt, Germany) and observed using a Leica SP2 confocal microscope (Leica Microsystems, Mannheim, Germany).

Cells were grown on the coverslips and fixed with 4% fresh paraformaldehyde for 10 min at room temperature, followed by blocking with 5% bovine serum albumin (BSA) at room temperature for 1 h. Next, cells were stained with FITC-Phalloidin for 1 h at room temperature in dark. After washing, cells were counterstained with DAPI for 10 min and mounted on 50% glycerol/PBS. A Leica SP2 confocal microscope (Leica Microsystems, Dresden, Germany) was used to observe the distribution of F-actin.

HEK293T cells were grown on glass coverslips and fixed in 4% paraformaldehyde for 15 min, washed with phosphate-buffered saline containing 20 mM glycine, permeabilized with the same buffer containing 0.05% Triton X-100, and successively washed with tris-buffered saline. Heteromers were detected using the Duolink II in situ PLA detection Kit (OLink; Bioscience, Uppsala, Sweden) following supplier’s instructions. A mixture of the primary antibodies (mouse anti-A_2AR antibody (1:100; 05-717, Millipore, Darmstadt, Germany; RRID:AB_309931) and rabbit anti-A₁R antibody (1:100; ab82477, Abcam, Bristol, UK; RRID: AB_2049141)) was used to detect A₁-A_2AHet together with PLA probes detecting mouse or rabbit antibodies. Then, samples were processed for ligation and amplification with a Detection Reagent Red and were mounted using a DAPI-containing mounting medium. Samples were analyzed in a Leica SP2 confocal microscope (Leica Microsystems, Mannheim, Germany) equipped with 405 nm and 561 nm laser lines. For each field of view, a stack of two channels (one per staining) and 4–6 Z-stacks with a step size of 1 μm were acquired. Images were opened and processed with Image J software (National Institutes of Health, Bethesda, MD, USA).