Imaging Particles Bound to Recombinant Proteins

Around 100–200 µg of MSU, CPPD, silica, cholesterol, and calcium carbonate crystals, or 1 x 10⁶S. cerevisiae particles were incubated either in human serum or HBSS at 37°C for 30 min. After washing with 5% BSA in HBSS, the particles were incubated with recombinant proteins as described above for flow cytometry. Fc-fusion proteins were stained the same way. Recombinant His-tagged proteins were detected using purified anti-His Tag (Biolegend, #362602, 5 µg/ml) and Alexa Fluor488 goat anti-mouse IgG (Biolegend, #405319, 5 µg/ml) binding at 4°C for 30 min each. Following a last washing step, the particles were mounted in Immunoselect Antifading Mounting Medium (Dianova, #SCR-038447).
Images were either acquired using an Olympus IX81 inverted microscope with a UPlanSApo 60x/1.35 Oil objective and Cell^R software (version 3.2; https://www.olympus-lifescience.com/en/software/) or a Zeiss 980 Airyscan 2 with an alpha Plan-Apochromat 63x/1.46 Oil Korr M27 objective in combination with Zeiss ZEN System software (blue edition version 3.0; www.zeiss.com/microscopy/int/products/microscope-software/zen.html). Brightness was adjusted, pseudo-color was inserted in the grayscale image, and scale bar was added using ImageJ (version 1.52d) (https://imagej.nih.gov/ij/) (25 (link)).