Biotinylated anti gl7 antibodies

Half kidneys were frozen in OCT and 5μm sections were prepared and stained using Texas Red-conjugated anti-mouse IgG (Invitrogen, Waltham, MA, USA) and fluorescein isothyocyanate (FITC)–conjugated anti-mouse C3 specific antibodies (Immunology Consultants Laboratory, Portland, OR, USA). Images were obtained using a 10x/0.3 Nikon Plan Fluor objective lens on a Keyence BZ-X710 Series fluorescence microscope. IgG deposition was quantified using Keyence BZ-X Viewer and Keyence BZ-X Analyzer software (Version 1.3.1.1, Keyence Corp., Osaka, Japan) and reported as arbitrary units representing average integrated brightness per glomerulus. Mean brightness of glomeruli per mouse were quantified by averaging integrated brightness of each glomerulus within a 10X field of view. Detection of GC B cells within spleens was done using FITC-conjugated anti-B220 antibodies, biotinylated anti-GL7 antibodies (EBiosciences), and Alexa Fluor 568-conjugated streptavidin (Invitrogen) on 5μm frozen sections. Mean area of GCs per mouse were quantified by averaging area of each GC within 10X field of view using microscope software mentioned above. All images to be compared were obtained using identical microscope settings (gain and exposure time).