Alexa fluor 488 goat anti mouse igg

For immunofluorescence staining, kidneys were snap-frozen in liquid nitrogen and stored at −20 °C until subsequent experiments. Kidney cryosections were fixed in ice-cold acetone for 5 min and air-dried. Sections were washed and blocked with 10% normal goat serum for one hr. Subsequently, the sections were stained with Alexa Fluor^® 488 goat anti-mouse IgG (BioLegend, San Diego, CA, USA) for 1 h at room temperature (RT). Tissue sections were mounted and examined by a conventional fluorescence microscope. The cell fluorescent intensity of IgG deposition in the sections was analyzed using the Image J software (National Institutes of Health, Bethesda, Maryland, USA). For pathological analysis, kidney tissues were fixed in 4% paraformaldehyde solution (PFA), processed into paraffin sections, and stained with hematoxylin and eosin (H&E) by Histopathology Services, Department of Pathology, HKU. Renal histologic abnormalities including glomerular, tubular, and vascular changes were assessed semi-quantitatively by two independent observers in a blinded manner. Scores were classified as 0 (no damage), 1 (mild damage, <25%), 2 (moderate damage, 25–50%), 3 (marked damage, >50–75%) or 4 (severe damage >75% of the total area) [19 (link),20 (link)]. At least 30 glomeruli were examined per mouse.

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)).