The ESB remains a single nuclear structure throughout S and G2 phases of the cell cycle. (A and B) Bloodstream form trypanosomes were subjected to double indirect 3D IF using polyclonal anti-TbRPA1 antibodies, monoclonal antibodies against the BB (BBA4), and DAPI staining. Cells of three different cell lines were grouped as defined by the number of nuclei (N), kinetoplasts (K), and BBs. 1K1N 2BB (n = 50) and 2K1N 2BB (n = 50) trypanosomes were scored for the number of extranucleolar pol I–containing ESBs. Representative 1K1N 2BB cells (A) and 2K1N 2BB (B) cells are shown as maximum intensity projections of three-channel 3D stacks showing anti–pol I signal in red, BBA4 signal in green, and DAPI staining in blue. (C) Maximum intensity projections of a two-channel 3D stack showing direct fluorescence of the fusion protein YFP-TbRPB5z in yellow and DAPI staining in blue. (D) Double indirect 3D IF of a cell line GFP tagged in the active VSG ES. Maximum intensity projections of a two-channel 3D stack showing polyclonal anti-TbRPA1 antibodies (red), monoclonal antibodies against GFP (green), and DAPI staining (blue) are shown. (A–D) Arrowheads indicate the ESB. Bars, 1 µm.