Immunocolocalization of clathrin heavy and light chains in Trypanosoma cruzi epimastigotes. Nucleus (arrowhead) and kinetoplast (arrow) DNA were stained with Hoechst 33342. (A–D): Transfected epimastigote expressing CLC-A/C incubated with TcCLC antiserum (A) and an antibody against protein A (B). (E–H): Transfected epimastigote expressing CLC-A/C incubated with monoclonal antibody against TcCHC (E) and an antibody against protein A (F). (I–L): Transfected T. cruzi epimastigote expressing Rab7-GFP (which localizes to the Golgi complex) (I), incubated with antiserum against TcCLC (J); note partial colocalization of the GFP and TcCLC signals. (M–P): CLC-A/C-expressing epimastigote incubated with Alexa Fluor 488-conjugated Albumin (M) and immunolabeled with antibody against protein A (N). Note colocalization at the cell anterior, in a region corresponding to the flagellar pocket. (Q–T): Wild type T. cruzi epimastigote immunolabeled with monoclonal antibody against TcCHC (Q) and incubated with Alexa Fluor 633-conjugated transferrin (R); Note absence of colocalization at the cell anterior, indicating that transferrin (red) localizes to the cytostome/cytopharynx and clathrin (green) to the flagellar pocket region. C, G, K, O &S: Merged images. D, H, L, P &T: Differential interference contrast (DIC) images of the parasite body. Scale bars = 5 μm.