Fluoview version 1.7c software

Double immunofluorescence staining was performed as previously described.^{36 (link)} Briefly, B cells were fixed in 4% paraformaldehyde (15 min, room temperature), washed with phosphate-buffered saline, permeabilized (0.1% Triton X-100 in phosphate-buffered saline, 15 min) and attached on precoated poly-L-lysine slides. Slides were blocked in 3% bovine serum albumin, followed by mouse monoclonal anti-NS3 (Abcam; 1:100, ab65407) or anti-core (ThermoFisher Scientific; 1:100, MA1-080) antibody staining. The cells were further incubated with rabbit polyclonal anti-CD19, anti-CD20 (Cell Signaling Technology, Inc., Danvers, MA, USA; 1:400; 3574), anti-CD20 (Abcam; 1:400; ab78237) or anti-B220 antibody (Biolegend; 1:200; #103201). Cells were washed with phosphate-buffered saline and incubated with goat anti-rabbit Alexa Fluor 488 and goat anti-mouse or anti-rat Alexa Fluor 597 (Invitrogen; 1:200). The stained slides were mounted using Vectashield mounting medium (Vector Laboratories Inc., Burlingame, CA, USA). Control slides were similarly processed, except primary antisera were omitted, which yielded no staining. Images were captured using an Olympus FV1000 confocal microscope and processed with Olympus Fluoview Version 1.7c software (Olympus Corporation, Tokyo, Japan).