Anti bcl6 antibody

Draining LNs and spleens were harvested and fixed with PLP buffer (0.05 M phosphate buffer containing 0.1 M l-lysine, pH 7.4, 2 mg/ml NaIO₄, and 10 mg/ml paraformaldehyde) for 12 h. After fixation, tissues were incubated in 30% sucrose for 6 h before embedding in optimum cutting temperature compound (Tissue-Tek). 30-μm sections were cut on a cryostat (CM3050S; Leica) and adhered to slides (Super Frost Plus Gold; Electron Microscopy Services). Frozen sections were permeabilized and blocked for 1–2 h in PBS containing 0.3% Triton X-100 (Sigma-Aldrich), 1% normal mouse serum, 1% BSA, and 10% normal goat serum. Sections were stained with directly conjugated antibodies for a minimum of 5 h at room temperature or 12 h at 4°C in a humidity chamber in the dark. Anti–mouse CD80, CD86, CD45.1, CD4, and B220 antibodies were purchased from BioLegends. Anti–mouse CD11c antibody was purchased from Thermo Fisher. Anti-Bcl6 antibody was purchased from BD Biosciences. Cell nuclei were visualized with JOJO-1 (Thermo Fisher). Stained slides were mounted with Fluoromount G (eBioscience) and sealed with a glass coverslip. Each section was visually inspected by epifluorescent light microscopy, and several representative sections from different lymphoid organs were acquired using a confocal microscope (SP8; Leica) and 40× objective (numerical aperture 1.3).