Duolink assay

HL-1 cells were seeded at 5.0 × 10⁴ cells/well on VWR Micro cover slips coated with gelatin/fibronectin in complete Claycomb medium, and incubated overnight at 37 °C with 5% CO₂. The following day, HL-1 cells were treated with 5 μM IWR-1 for 24–48 hours. MiRCURY LNA™ Detection probe was used to stain for miR-133a or scramble, and tyramide signal amplification (TSA) system (Life technologies) was used to detect miRNA or scramble signal, while nuclei were counterstained with 4′,6-diamidino-2-phenylindole, dihydrochloride (Life Technologies). Fluorescent images were taken with a laser scanning confocal microscope (Olympus, FV1000/SIMS). For he AGO2-DNMT3B interaction, the Duo-Link assay (Sigma Aldrich) was used following the manufacturer’s instructions.

MUSE cells were grown on cover slides in MW24 plate and then fixed in 4% formaldehyde solution for 15 min at room temperature. We performed Duolink assay following the manufacturer's instructions (Sigma) using SSEA‐3 (IBL) and FGF2 (Elabscience) as primary antibody. Micrographs were taken under a fluorescence microscope (Leica). The percentage of positive cells was calculated by counting at least 300 cells in different microscope fields.

Dually tagged parasites syringe-released from host cells washed twice in cold PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. After fixation, cells were washed once in PBS and then seeded in poly-L-lysine (Sigma-Aldrich) coated glass coverslips. The cells were permeabilized using PBS plus 0.25% Triton X-100 for 30 min at RT. DuoLink^® assay (Sigma-Aldrich) was performed according to the manufacturer's instructions with the following modifications: overnight blocking in a humidity chamber and five washes with 1 ml washing buffer per coverslip.

MSCs were cultured on cover slides in a 24 multi-wells plate. The Duolink assay was performed following the manufacturer's instructions (Sigma Aldrich, MO, USA) using His-Tag (Elabscience, TX, USA) and CAV1 (Elabscience, TX, USA), or RARα (E-AB-93398, Elabscience, TX, USA) as primary antibodies. Micrographs were captured under a fluorescence microscope (Leica, Germany).

The Duolink assay (Sigma-Aldrich) was used to study if exogenous C1q promotes molecular association of LAIR-1 and CD33 on the cell surface. Freshly isolated monocytes were either untreated or treated with C1q (20 ug/ml) for 40 min; resuspended in PBS, fixed in paraformaldehyde and then deposited onto slides by cytocentrifugation (Cytospin, Shandon). After staining with primary Abs to CD33 and LAIR-1 of distinct species, species-specific secondary antibodies containing complementary DNA probes were added followed by enzymatic ligation and rolling circle amplification using fluorescently labeled complementary probes. Nuclear counterstaining was performed using DAPI; red fluorescent spots representing protein-protein interaction complexes were detected and visualized using an Axio Image. Z1 ApoTome enabled microscope (Zeiss).