Fm1000d confocal laser scanning microscope

Frozen kidney sections were incubated with FITC-anti-IgG (Southern Biotech Birmingham) or FITC-anti-C3 (MP Biomedicals) and then mounted with Fluoromount/Plus (Diagnostic BioSystems). All samples were visualized using a FM1000D confocal laser scanning microscope (Olympus), and images were captured and analyzed using the FV10-ASW viewer (Olympus). The mean fluorescence intensity (MFI) of FITC was calculated using ImageJ software.

Mouse kidneys were embedded in Tissue Tek embedding medium for frozen tissue blocks (Sakura Finetechnical Co., Ltd., Tokyo, Japan). Frozen kidney sections were sectioned and incubated with FITC-conjugated anti-mouse IgG antibody (Southern Biotech Birmingham, UK). Stained samples were mounted with Fluoromount/Plus (Diagnosis Biosystems, CA, USA). All samples were visualized using an FM1000D confocal laser scanning microscope (Olympus, Tokyo, Japan), and the images were analyzed using the FV10-ASW viewer (Olympus) or Image J software (Schneider, Nat. Methods 2012). For the IHC of color development, frozen kidney sections were incubated with anti-mouse CD11b (clone M1/70), anti-mouse CD16.2 (FcγRIV) (clone 9E9), anti-mouse TREML4 (clone 16E5), and anti-mouse Ly-6G (clone 1A8). Samples were mounted with Fluoromount/Plus (Diagnosis Biosystems) and analyzed using an EVOS microscope (Thermo Fisher Scientific) or a BZ-X710 fluorescence microscope (Keyence, Osaka, Japan). The number of glomeruli assessed was 5 to 10 from an individual kidney in B6 WT (n = 4 or 5), saline (n = 5), control IgG1 (n = 6), and anti-TLR7 (n = 5). The dots show the average percentage of the indicated positive staining area. Ratios (%) of the indicated positive staining area to the total area of one glomerulus were calculated using the BZ-X710 fluorescence microscope software.