For IHC, kidneys were fixed with 10% buffered formaldehyde and embedded in paraffin. Slices of 4 μm thick were stained with H&E or periodic acid–Schiff (PAS) and observed on an EVOS microscope (Thermo Fisher Scientific). The slides were scored blindly by an expert with more than 10 years of experience with renal histopathology, as described previously (62 (link)). Briefly, severity of GN was assessed by mild to moderate increase in mesangial cellularity, thickening of the GBM, endocapillary hypercellularity, and crescents formation. The tubulointerstitial inflammation was measured by assessing tubular atrophy and tubulointerstitial inflammation.
Kidneys were perfused with PBS containing EDTA (MilliporeSigma) before harvesting. Kidneys were digested at 37°C in 1 mg/mL collagenase IV (Worthington) in complete RPMI for 30 minutes, filtered through 70 mm strainers, and washed twice in PBS. For flow cytometry, the following antibodies were used: CD45 (30-F11, eBioscience), CD3 (145-2C11, BioLegend), CD4 (GK1.5, BioLegend), CD8 (53-6.7, BioLegend), CD45R/B220 (RA3-6B2, BioLegend), Ly6G (IA8, eBioscience), CD11b (M1/70, BioLegend), F4/80 (BM8, BioLegend), and Ly6C (HK1.4, eBioscience). Samples were acquired on BD LSRFortessa cytometer (BD Biosciences) and analyzed by FlowJo software (Tree Star Inc.).
Kidneys were perfused with PBS containing EDTA (MilliporeSigma) before harvesting. Kidneys were digested at 37°C in 1 mg/mL collagenase IV (Worthington) in complete RPMI for 30 minutes, filtered through 70 mm strainers, and washed twice in PBS. For flow cytometry, the following antibodies were used: CD45 (30-F11, eBioscience), CD3 (145-2C11, BioLegend), CD4 (GK1.5, BioLegend), CD8 (53-6.7, BioLegend), CD45R/B220 (RA3-6B2, BioLegend), Ly6G (IA8, eBioscience), CD11b (M1/70, BioLegend), F4/80 (BM8, BioLegend), and Ly6C (HK1.4, eBioscience). Samples were acquired on BD LSRFortessa cytometer (BD Biosciences) and analyzed by FlowJo software (Tree Star Inc.).