Spleens were embedded in OCT compound (SAKURA) and frozen at −80°C. 5 μm sections were prepared using a cryo‐microtome (Reichert‐Jung 2800 Frigocut) with a S35 knife (Feather) and fixed on SuperFrost Plus slides (Thermo Scientific) by treatment with pure acetone.
For generating overviews of spleens, sections were rehydrated with PBS + 2% BSA + 0.1% Na‐azide and blocked with Fc‐Block (α‐CD16/32; BD Biosciences).
For detection of germinal centers, spleens were fixed in 4% paraformaldehyde (PFA, Santa Cruz) for 3–4 h at 4°C and subsequently dehydrated in 30% sucrose. Sections were fixed with (95:5) acetone/methanol (Merck) and afterwards blocked with 2.5% rabbit and 2.5% mouse serum (vector laboratories). Sections were stained with antibodies enlisted inAppendix Table S9 and mounted with fluoromount‐G containing 4′,6′‐diamidino‐2‐phenylindole (DAPI; Southern Biotech). Stained sections were analyzed using fluorescence microscopes Axioskop 2 (Zeiss) or DMi8 (Leica).
For generating overviews of spleens, sections were rehydrated with PBS + 2% BSA + 0.1% Na‐azide and blocked with Fc‐Block (α‐CD16/32; BD Biosciences).
For detection of germinal centers, spleens were fixed in 4% paraformaldehyde (PFA, Santa Cruz) for 3–4 h at 4°C and subsequently dehydrated in 30% sucrose. Sections were fixed with (95:5) acetone/methanol (Merck) and afterwards blocked with 2.5% rabbit and 2.5% mouse serum (vector laboratories). Sections were stained with antibodies enlisted in