Staining for intracellular AID protein by flow cytometry.

CD19⁺ B cells in PBMCs or purified B cells were stained with a viability dye for 30 minutes at room temperature (Fixable Viability Dye eFluor 520, eBioscience, San Diego, CA), washed, stained with surface B cell markers as above for 20 minutes at room temperature, fixed with fixation medium (Medium A, Invitrogen) at room temperature for 10 minutes, washed and permeabilized with 5% methanol on ice for 10 minutes, which allowed optimal permeabilization and recognition of intracellular AID without affecting surface antigen expression. After washing with FACS buffer, cells were stained with rat anti-AID antibody (1:333; EK2 5G9, Cell Signaling, Danvers, MA) for 30 minutes at room temperature, washed, detected with anti-rat IgG_2b-PE (1:100; R2B-7C3, eBioscience) for 20 minutes on ice, washed twice with FACS buffer then data was acquired using flow cytometry. Results with CD3+ T cells and secondary anti-rat IgG alone served as negative controls.