Fluorescence microscopy

To visualize Sirt1 and AID expression in B cells, cells were spun onto glass slides (800 rpm for 5 min; Cytospin 4, Thermo Fisher Scientific) and then fixed with 3% paraformaldehyde in 250 mM Hepes (pH 7.4), stained with APC-labeled anti-Sirt1 Ab (as above) or Alexa Fluor 647–anti-AID Ab (bs-7855R-A647, Bioss), and mounted in ProLong Gold Antifade Reagent with DAPI (4′,6-diamidino-2-phenylindole; Invitrogen). Fluorescent images were captured using a 10× objective lens with a Zeiss Axio Imager Z1 fluorescence microscope.

To analyze IgM- and IgA-producing cells, intestinal sections were heated at 80°C to adhere to glass slides, washed four times in xylene for 2 min, dehydrated twice with 100% ethanol for 1 min, twice with 95% ethanol for 1 min, and washed twice in water for 1 min. Antigens were unmasked using 2 mM EDTA in 100°C for 40 min followed by a cooling step at 25°C, washed three times with tris-buffered saline (TBS), and blocked using 10% BSA for 15 min. Slides were again washed three times with TBS and stained with rabbit anti-IgA Ab (PA-1-30826, Thermo Fisher Scientific) followed by Alexa Fluor 488–conjugated anti-rabbit IgG (H+L) Ab F(ab′)₂ fragment (4414, Cell Signaling Technology) and PE-conjugated goat-anti mouse-IgM mAb (clone RMM-1; BioLegend) for 2 hours in a moist dark chamber. After washing three times with Triton X-100 (0.1%) in TBS, slides were air-dried, and coverslips were mounted with ProLong Gold Antifade Reagent using DAPI (Invitrogen). To analyze germinal center structure, 10-μm spleen sections were prepared by cryostat and loaded onto positively charged slides, fixed in cold acetone and stained with PE-GL7 and FITC-B220 mAb for 1 hour at 25°C in a moist chamber. Coverslips were mounted using ProLong Gold Antifade Reagent with DAPI for microscopy analysis. All the Abs and mAbs used in the above experiments are listed in table S1A.