Visualizing Epitope-Tagged Mitosomal Proteins

E. histolytica transformants expressing epitope-tagged mitosomal proteins were previously established [5] (link). Approximately 5×10⁵ trophozoites were resuspended in 2 ml BI-S-33 medium and seeded onto a molybdenum disk (Nissin EM Co., JAPAN) in a well of a 24-well plate. After 15-min incubation at 35.5°C, the molybdenum disk that amoebas adhered to was removed and immediately immersed in liquid propane at −175°C. The disk was further fixed and sectioned as previously described [21] (link). The disk was reacted with primary antibody diluted at 1∶2000 (anti-Cpn60 antiserum) and 1∶500 (anti-HA monoclonal antibody) in phosphate-buffered saline containing 1.5% bovine serum albumin for overnight at 4°C. The samples were then reacted with colloidal gold-conjugated anti-rabbit or anti-mouse secondary antibody (1∶20) for 1 h at room temperature. Samples were examined by electron microscopy at Tokaii Microscopy., Inc (Nagoya, JAPAN).