Immunofluorescence staining.

Six-micrometer-thick sagittal sections from frozen, OCT-embedded tissues were warmed to room temperature, fixed in absolute acetone, and washed twice with PBS. Nonspecific binding and endogenous Fc receptors were blocked with 1% BSA and 5% heat-inactivated goat/rat/rabbit serum diluted in PBS for 1 hour at room temperature. Antibodies were diluted in PBS containing 2% heat-inactivated fetal bovine serum (HI-FBS) and 1% BSA, applied to blocked tissue sections, and incubated overnight at 4°C in a humidified chamber. Nuclei were stained with DAPI, washed in cold PBS, and mounted with a 1:1 solution of PBS and glycerol. Kidney autofluorescence was blocked with 0.05% Sudan Black B in 70% ethanol for 15 minutes at room temperature, washed twice in deionized water (10 minutes each), and mounted as described above. Slides were sealed and imaged in a blinded fashion with either an Olympus FV1000 FCS confocal (at ×100, ×200, or ×600 original magnification) or a Zeiss Axio Observer D1 epifluorescent microscope (×100, ×200, or ×400 magnification). A minimum of 10 fields of view (FOV) were captured per sample. Images were converted from binary data format with either Olympus FV-10ASW (version 04.02.02.09) or Zeiss Zen Blue (version 2.6) software. Representative isotype images are shown in Supplemental Figure 1, B–H. Image quantification was performed according to methods described in Oil Red O staining.