Scanning electron microscopy (SEM)
For SEM experiments, antigen coated coverslips were used. Sterile
coverslip were coated in 24-well plates with 60 μl poly-D-lysine
solution (50 μg/ml) (C0312, Beyotime) overnight at 4 °C. The next day,
the coverslips were washed with sterile PBS, dried, and incubated with
100 μl 10 μg/ml F(ab’)2 antibody at 37 °C for 3 h. Purified B cells at a
concentration of 3 × 106 cells/ml were added gently
onto the antigen coated coverslips and stimulated at 37 °C for 10 min. A
total of 400 μl of 2.5% glutaraldehyde was used to terminate the
stimulation and fix the cells for 20 min (on ice). Scanning electron
micrographs were captured using a Ultra-high Resolution Scanning
Electron Microscope (SU8010,
HITACHI, Tokyo, Japan). Cellular filopodia expansion was imaged and the
number and length of filopodia were quantified using ImageJ software
(NIH, Bethesda, MD, USA).