Intracellular Signaling Molecules in Activated B Cells

Intracellular immunofluorescence staining of the signalling molecule in the IS of activated B cells were performed according to our published protocols7 (link)15 (link)32 (link). Briefly, B cells were fixed with 4% PFA fixation for 30 min at RT. After washed with 1 × PBS, B cells were permeabilized with 0.2% Triton X-100 for 20 min and then blocked with 100 μg ml⁻¹ goat non-specific IgG (Jackson ImmunoResearch Laboratory, 005-000-003) for 1 h at RT. Subsequently, cells were incubated with various primary antibodies including Anti-phospho-Syk (pY525/526) Ab (Cell Signaling, #2711, 1:500 dilution), Anti-phospho-PI3K p85 (pY458)/p55 (pY199) Ab (Cell Signaling, #4228, 1:500 dilution) and Anti-phospho-BLNK Ab (Biodragon, 1:500 dilution) at 37 °C for 1 h and then Alexa Fluor 568 conjugated F(ab)₂ fragment goat anti- rabbit IgG (Invitrogen, 1:2,000 dilution) was used as the secondary antibody. TIRFM Images were processed with Image J (NIH, USA)7 (link)8 (link)15 (link).