Clone 2 41

Excised kidneys we embedded in OCT compound, snap frozen in liquid nitrogen and 10uM sections cut on a cryostat. After air drying, sections were fixed in 4% paraformaldehyde for 30 minutes at room temperature (RT) and then rinsed before staining. For immunofluorescence staining, sections were blocked with 5% goat serum in PBS for 30 minutes at RT, then incubated with directly-labeled antibodies diluted in TBST+BSA for one hour at RT and rinsed 3 times with PBST. APC-labelled goat anti-mouse IgG was from Jackson ImmunoResearch (cat#115-135-164) and FITC-labeled rat anti-mouse IgM was from BD Bioscience (clone II/41; Cat#553437). Slides were mounted with ProLong gold antifade solution (Invitrogen #P36935) and images captured using an EVOS cell imaging system (ThermoFisher Scientific). Images were analyzed using Fiji software as follows: lines were drawn across the maximum diameter of each individual glomeruli and the Measure function was used to determine the average fluorescence intensity and length (as an estimate of glomeruli size).

Cytospins of isolated splenic IgM+ B cells and spleen cryosections were prepared as described previously [36 (link)]. Cytospins and cryosections were fixed in acetone solution (10 min, -20°C). The alkaline phosphatase anti-alkaline phosphatase (APAAP) technique was used to phenotype IgM+ (clone II/41; 1:10; BD Biosciences) B cells [37 (link)]. After incubation with the primary antibody slides were washed with TBS-Tween (0.05% Tween 20, Serva, Heidelberg, Germany) and incubated with the bridging antibody (rabbit anti-rat, 1:100 Dako, Hamburg, Germany) followed by the APAAP complex (Dako). As a substrate for the alkaline phosphatase Fast blue (Sigma) was used. All sections were counterstained with hemalaun and mounted in glycergel (Dako). BrdU⁺ cells were stained using peroxidase conjugated mAbs (clone BMG-6H8; 1:25; Roche Diagnostics, Mannheim, Germany) and visualized by diaminobenzidine substrate kit (Vector Laboratories, Burlingame, USA).