N sim microscopy

CH12F3 cells were cultured in a 6‐well plate at 0.5 million/mL overnight under the stimulation of anti‐CD40, IL4, and TGF‐β. Cytokine‐stimulated CH12F3 cells were nucleofected with 1 μg AID plasmid and 2 μg FBL‐mCherry plasmid per million cells following the instructions of Lonza nucleofection kits (Lonza #V4XC‐2012). Then the cells were fixed with 4% formaldehyde for 15 min at room temperature. PBS with 0.5% Triton X‐100 was used for permeabilization after triple PBS wash for about 15 min. Cells were then blocked with PBS with 3% BSA for 45 min at room temperature, followed by a primary antibody of AID#c (A16217; Abclonal; epitope: 185–198 aa) incubation for 2 h. After a 4‐step wash with the PBS and 0.2% Tween‐20, the cells were incubated with the secondary antibody Alxa Fluor™ 647 goat anti‐rabbit IgG (Invitrogen, #1959073) for 1 h. Then the cells were washed and mounted to a microscope slide for microscopy analysis.
Cells were imaged with the Nikon N‐SIM microscopy at a setting as: pixel—0.12 μm, laser—640 nm, intensity—65%, time resolution—100 ms per frame; laser—561 nm, intensity—70%, time resolution—60 ms; the laser—405 nm, intensity—40%, time resolution—30 ms. The images were taken through the thin slice scan technology. The N‐SIM software package was used to process multiple images.

Glass coverslips with cells after IF staining were mounted on PVA (polyvinyl alcohol, Sigma-Aldrich, St. Louis, MO, USA). Samples were examined using N-SIM microscopy (Nikon) with an EMCCD camera (iXon 897, AndorT). Then, 488 and 561 nm laser excitation and 100x/1.49 NA oil immersion objectives were used. Exposure conditions were adjusted to get a typical yield of about 5000 max counts (16-bit raw image) for minimal bleaching. Images have been captured as serial optical sections of the same cell in z-axis with 0.12 μm z-steps. For serial images in wide-field mode, the AutoQuant blind deconvolution algorithm was used. Image analysis, visualization, archiving, and volume views were performed using NIS-Elements 4.2 microscope imaging software (Nikon).

Cells were cultured on coverslips to the appropriate density, fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.2% Triton X-100. The slides were then blocked in 1% bovine serum albumin (BSA) for 30 min, incubated with the appropriate primary antibodies diluted in 1 % BSA for 1.5 h, washed with PBS, and incubated with secondary antibodies (1:500 dilution) for 1 h. The slides were then washed, mounted, and observed by Nikon N-SIM microscopy. For confocal images, Zeiss 780 and 800 confocal microscopies were employed. All antibodies for immunofluorescence staining were listed in Table S3.